Dry growth hormone composition transiently linked to a polymer carrier

ABSTRACT

The present invention relates to dry compositions of rhGH polymer prodrug containing a lyoprotectant and, optionally, one or more than one excipient. Such compositions are stable for at least 1 year, when stored at 2-8° C. The invention further relates to methods of manufacturing said compositions, containers comprising such composition as well as a kit of parts.

The present application is a continuation of U.S. patent applicationSer. No. 15/340,595 filed on Nov. 1, 2016, which is a continuation ofU.S. patent application Ser. No. 13/515,621 filed on Jun. 13, 2013, nowU.S. Pat. No. 9,511,122, which claims priority from PCT PatentApplication No. PCT/EP2010/069710 filed on Dec. 15, 2010, which claimspriority from European Patent Application No. EP 09179335.6 filed onDec. 15, 2009, the disclosures of which are incorporated herein byreference in their entirety.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates to dry compositions of rhGH polymerprodrugs, methods for their manufacture as well as containers and kitsof parts comprising said compositions.

It is noted that citation or identification of any document in thisapplication is not an admission that such document is available as priorart to the present invention.

Growth hormone (GH) is a hormone that stimulates growth and cellreproduction in humans and other animals. It is a 191-amino acid,single-chain polypeptide hormone which is synthesized, stored, andsecreted by the somatotroph cells within the lateral wings of theanterior pituitary gland. The hormone is also known as somatotropin whenreferring to human growth hormone (hGH) produced by recombinant DNAtechnology, and is abbreviated “rhGH”.

Growth hormone has a variety of functions in the body, the mostnoticeable of which is the increase of height throughout childhood, andthere are several diseases which can be treated through the therapeuticuse of GH.

Growth hormone deficiency is caused by insufficient production of growthhormone which causes various negative effects. During infancy andchildhood the most prominent feature is growth failure, resulting inshort stature, whereas in adults it causes diminished lean body mass,poor bone density and other physical and psychological effects. Thestandard treatment of growth hormone deficiency is daily injections withrecombinant human growth hormone (rhGH) under the skin or into themuscle.

To free the pediatric patient group from the burden of daily injections,long-acting growth hormone compositions are intended to provide therapyof growth hormone deficiency in children. Various compositions oflong-acting or sustained release human growth hormone are described.Growth hormone depots of the first generation were based on slow-releasecompositions of the human growth hormone in highly viscous liquid suchas sucrose acetate isobutyrate (WO 01/78683, Genentech) or biodegradablePLGA (polylactide/polyglycolide) gel. U.S. Pat. No. 5,645,010, Alkermes,describes a composition of zinc-complexed hGH in PLGA. A correspondingmarketed product (Nutropin Depot) was offered as single-dose injectablecomposition. For various reasons, Nutropin Depot did not showsignificant market uptake and was discontinued.

More recent polymer-based compositions employed hyaluronic acid insteadof PLGA (US 2008/0063727, LG Life Sciences). Other developments focusedon PEG conjugates of growth hormone with the goal of extending both theabsorption phase from the subcutaneous tissue after injection as well asthe terminal half-life of the circulating conjugate (U.S. Pat. No.4,179,337 describes the PEGylation of somatropin, as does WO 03/044056,Pharmacia, and WO 06/102659, Nektar).

Nevertheless, only few details are available on how to formulatePEGylated growth hormone. As PEG itself is a highly viscous material,corresponding protein conjugates carrying high molecular weight PEGchains also exhibit strongly enhanced viscosity as compared to theunmodified protein. This situation is even more pronounced as it isdesirable to provide for sufficient material for a less frequent thandaily dosage while at the same time seeking to minimize the injectionvolume. Consequently, compositions of long-acting PEGylated growthhormone are much more concentrated and viscous than the existingonce-daily compositions of unmodified growth hormone.

WO 2006/071840, Ambrx, details compositions of PEGylated growth hormonevariants incorporating non-natural amino acids. WO 2007/025988, Novo,describes compositions containing growth hormone and PEG through anoxime bond. US 26/257479, Novo, details compositions of PEG growthhormone conjugates in PLGA. An expansion of the scope of PEGylation wasrecently introduced by the use of reversible linkers in a prodrugapproach. A PEGylated prodrug of hGH exhibits significantly reducedbioactivity of the PEGylated conjugate but exhibits full uncompromisedbioactivity of the free growth hormone released from the conjugate.

Compositions of such hGH prodrugs do not only have to take into accountthe viscosity introduced by the PEG component, but also have to providefor sufficient prodrug stability to avoid premature hGH release duringstorage. In case the reversible linkage between the PEG moiety and thedrug is degraded during storage, the concentration of readily availabledrug is increased, which leads to the risk of overdosage. In addition,any drug released during storage is subject to rapid renal clearanceupon application to a patient and, as a consequence, the time for whichthe long-acting composition provides therapeutically relevant amounts ofdrug is reduced. This poses the risk of unmet medical needs.

Furthermore, it is known that rhGH will undergo decomposition reactionsunder storage conditions, leading to impurities of the correspondingcomposition. It is therefore mandatory to identify suitable rhGH polymerprodrug compositions, wherein the rhGH will exhibit an acceptableimpurity profile.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

It is further noted that the invention does not intend to encompasswithin the scope of the invention any previously disclosed product,process of making the product or method of using the product, whichmeets the written description and enablement requirements of the USPTO(35 U.S.C. 112, first paragraph) or the EPO (Article 83 of the EPC),such that applicant(s) reserve the right to disclaim, and herebydisclose a disclaimer of, any previously described product, method ofmaking the product, or process of using the product.

SUMMARY OF THE INVENTION

It is therefore of paramount interest to develop compositions of hGHpolymer prodrugs that ensure the stability of the rhGH polymer prodrugcompound.

Furthermore, it is desirable to provide for single-dose as well asmultiple-dose compositions of such hGH polymer prodrugs.

Thus an object of the present invention is to provide such compositions.

The object is achieved by a dry composition comprising a therapeuticallyeffective amount of a rhGH polymer prodrug and one or morelyoprotectants and optionally one or more pharmaceutically acceptableexcipients, wherein the growth hormone is transiently linked to apolymer carrier.

In the context of the present invention terms and phrases are used asfollows.

Since the recombinant human GH is identical in sequence to natural humanGH, the term recombinant human growth hormone (rhGH) relates herein alsoto so-called biogenerics equivalents. Thus, the terms rhGH and hGH canbe used synonymously within the meaning of the present invention.

As known to the person skilled in the art, it is today routine work tomake e.g. minor amino changes of a biologics of interest (herein: GH)without significantly affecting the activity of the biologics.

Besides recombinant human and biogenerics, the term recombinant humangrowth hormone relates herein also to all possible rhGH polypeptides.

A precise description of possible rhGH polypeptides is given in WO-A2005/079838 from the Pharmacia Corporation provided on page 15,paragraph 0043 till and including paragraph 0053.

The term “hGH polypeptide or hGH protein”, when used herein, encompassesall hGH polypeptides, preferably from mammalian species, more preferablyfrom human and murine species, as well as their variants, analogs,orthologs, homologs, and derivatives, and fragments thereof that arecharacterized by promoting growth in the growing phase and inmaintaining normal body composition, anabolism, and lipid metabolism.Optionally, the term “hGH polypeptide or hGH protein” further includeshGH with one or more additional amino acid residues compared tonaturally occurring hGH variants, whereas these additional amino acidsresidues may either at the N-terminus, C-terminus and/or internally. Itis understood that the term “hGH polypeptide or hGH protein” also refersto hGH variants with any combination of additional, deleted or exchangedamino acid residues.

Preferably, the term “hGH polypeptide or hGH protein”, when used herein,encompasses all hGH polypeptides, preferably from mammalian species,more preferably from human and murine species, as well as theirvariants, analogs, orthologs, homologs, and derivatives, and fragmentsthereof that are characterized by promoting growth in the growing phaseand in maintaining normal body composition, anabolism, and lipidmetabolism.

More preferably, the hGH polypeptide or hGH protein is at least 95%identical to the sequence using the 1-letter code for amino acidsaccording to IUPAC-IUB known to the person skilled in the art givenbelow:

(SEQ ID NO: 1) FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKFQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF

The term “hGH polypeptide or protein” preferably refers to the 22 kDahGH polypeptide having a sequence as disclosed in A. L. Grigorian etal., Protein Science (2005), 14, 902-913 as well as its variants,homologs and derivatives exhibiting essentially the same biologicalactivity (promoting growth in the growing phase and in maintainingnormal body composition, anabolism, and lipid metabolism). Morepreferably, the term “hGH polypeptide or protein” refers to thepolypeptide having exactly the abovementioned sequence.

The term “hGH polypeptide variants”, as used herein, refers topolypeptides from the same species but differing from a reference hGHpolypeptide. Generally, differences are limited so that the amino acidsequences of the reference and the variant are closely similar overalland, in many regions, identical. Preferably, hGH polypeptides are atleast 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to a referencehGH polypeptide, preferably the hGH polypeptide having a sequence asindicated in A. L. Grigorian et al., Protein Science (2005), 14,902-913. By a polypeptide having an amino acid sequence at least, forexample, 95% “identical” to a query amino acid sequence, it is intendedthat the amino acid sequence of the subject polypeptide is identical tothe query sequence except that the subject polypeptide sequence mayinclude up to five amino acid alterations per each 100 amino acids ofthe query amino acid sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence. The query sequence may be an entire amino acidsequence of the reference sequence or any fragment specified asdescribed herein.

Such hGH polypeptide variants may be naturally occurring variants, suchas naturally occurring allelic variants encoded by one of severalalternate forms of a hGH occupying a given locus on a chromosome of anorganism, or isoforms encoded by naturally occurring splice variantsoriginating from a single primary transcript. Alternatively, a hGHpolypeptide variant may be a variant that is not known to occurnaturally and that can be made using art-known mutagenesis techniques.

It is known in the art that one or more amino acids may be deleted fromthe N-terminus or C-terminus of a bioactive peptide or protein withoutsubstantial loss of biological function (see for instance, Ron et al.,(1993), Biol. Chem., 268 2984-2988 which disclosure is herebyincorporated by reference in its entirety).

It also will be recognized by one of ordinary skill in the art that someamino acid sequences of hGH polypeptides can be varied withoutsignificant effect of the structure or function of the protein. Suchmutants include deletions, insertions, inversions, repeats, andsubstitutions selected according to general rules known in the art so asto have little effect on activity. For example, guidance concerning howto make phenotypically silent amino acid substitutions is provided inBowie et al. (1990), Science 247:1306-1310, hereby incorporated byreference in its entirety, wherein the authors indicate that there aretwo main approaches for studying the tolerance of an amino acid sequenceto change.

The first method relies on the process of evolution, in which mutationsare either accepted or rejected by natural selection. The secondapproach uses genetic engineering to introduce amino acid changes atspecific positions of a cloned hGH and selections or screens to identifysequences that maintain functionality. These studies have revealed thatproteins are surprisingly tolerant of amino acid substitutions. Theauthors further indicate which amino acid changes are likely to bepermissive at a certain position of the protein. For example, mostburied amino acid residues require nonpolar side chains, whereas fewfeatures of surface side chains are generally conserved. Other suchphenotypically silent substitutions are described in Bowie et al.,(1990) supra, and the references cited therein.

Typically seen as conservative substitutions are the replacements, onefor another, among the aliphatic amino acids Ala, Val, Leu and Pheinterchange of the hydroxyl residues Ser and Thr, exchange of the acidicresidues Asp and Glu, substitution between the amide residues Asn andGln, exchange of the basic residues Lys and Arg and replacements amongthe aromatic residues Phe, Tyr. In addition, the following groups ofamino acids generally represent equivalent changes: (1) Ala, Pro, Gly,Glu, Asp, Gln, Asn, Ser, Thr; (2) Cys, Ser, Tyr, Thr; (3) Val, He, Leu,Met, Ala, Phe; (4) Lys, Arg, His; (5) Phe, Tyr, Trp, His.

The term hGH polypeptide also encompasses all hGH polypeptides encodedby hGH analogs, orthologs, and/or species homologues. As used herein,the term “hGH analogs” refers to hGHs of different and unrelatedorganisms which perform the same functions in each organism but whichdid not originate from an ancestral structure that the organisms'ancestors had in common. Instead, analogous hGHs arose separately andthen later evolved to perform the same function (or similar functions).In other words, analogous hGH polypeptides are polypeptides with quitedifferent amino acid sequences but that perform the same biologicalactivity, namely promoting growth in the growing phase and inmaintaining normal body composition, anabolism, and lipid metabolism. Asused herein, the term “hGH orthologs” refers to hGHs within twodifferent species which sequences are related to each other via a commonhomologous hGH in an ancestral species but which have evolved to becomedifferent from each other. As used herein, the term “hGH homologs”refers to hGHs of different organisms which perform the same functionsin each organism and which originate from an ancestral structure thatthe organisms' ancestors had in common. In other words, homologous hGHpolypeptides are polypeptides with quite similar amino acid sequencesthat perform the same biological activity, namely promoting growth inthe growing phase and in maintaining normal body composition, anabolism,and lipid metabolism. Preferably, hGH polypeptide homologs may bedefined as polypeptides exhibiting at least 40%, 50%, 60%, 70%, 80%,90%, 95%, 96%, 97%, 98% or 99% A identity to a reference hGHpolypeptide, preferably the hGH polypeptide having a sequence asmentioned above.

Thus, a hGH polypeptide may be, for example: (i) one in which one ormore of the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue mayor may not be oneencoded by the genetic code: or (ii) one in which one or more of theamino acid residues includes a substituent group: or (iii) one in whichthe hGH polypeptide is fused with another compound, such as a compoundto increase the half-life of the polypeptide (for example, polyethyleneglycol): or (iv) one in which the additional amino acids are fused tothe above form of the polypeptide, such as an IgG Fc fusion regionpeptide or leader or secretory sequence or a sequence which is employedfor purification of the above form of the polypeptide or a pro-proteinsequence.

hGH polypeptides may be monomers or multimers. Multimers may be dimers,trimers, tetramers or multimers comprising at least five monomericpolypeptide units. Multimers may also be homodimers or heterodimers.Multimers may be the result of hydrophobic, hydrophilic, ionic and/orcovalent associations and/or may be indirectly linked, by for example,liposome formation. In one example, covalent associations are betweenthe heterologous sequences contained in a fusion protein containing ahGH polypeptide or fragment thereof (see, e.g., U.S. Pat. No. 5,478,925,which disclosure is hereby incorporated by reference in its entirety).In another example, a hGH polypeptide or fragment thereof is joined toone or more polypeptides that may be either hGH polypeptides orheterologous polypeptides through peptide linkers such as thosedescribed in U.S. Pat. No. 5,073,627 (hereby incorporated by reference).

Another method for preparing multimer hGH polypeptides involves use ofhGH polypeptides fused to a leucine zipper or isoleucine zipperpolypeptide sequence known to promote multimerization of the proteins inwhich they are found using techniques known to those skilled in the art,including the teachings of WO 94/10308. In another example, hGHpolypeptides may be associated by interactions between Flag® polypeptidesequence contained in fusion hGH polypeptides containing Flag®polypeptide sequence. hGH multimers may also be generated using chemicaltechniques known in the art such as cross-linking using linker moleculesand linker molecule length optimization techniques known in the art(see, e.g., U.S. Pat. No. 5,478,925), techniques known in the art toform one or more inter-molecule cross-links between the cysteineresidues located within the sequence of the polypeptides desired to becontained in the multimer (see, e.g., U.S. Pat. No. 5,478,925, additionof cysteine or biotin to the C terminus or N-terminus of hGH polypeptideand techniques to generate multimers containing one or more of thesemodified polypeptides (see, e.g., U.S. Pat. No. 5,478,925), or any ofthe 30 techniques to generate liposomes containing hGH multimers (see,e.g., U.S. Pat. No. 5,478,925), which disclosures are incorporated byreference in their entireties.

As used herein, the term “hGH polypeptide fragment” refers to anypeptide or polypeptide comprising a contiguous span of a part of theamino acid sequence of an hGH polypeptide, preferably the polypeptidehaving the above-mentioned sequence, i.e. it refers to hGH polypeptideshaving one or more deletions of at least one amino acid residue at theN-terminus, C-terminus and/or internally, when compared to naturallyoccurring variants.

It is well known that rhGH or its variants, conjugates or derivativesmay undergo decomposition reactions which may lead to impurities, suchas:

-   -   Succinimide and isoaspartate degradation products (impurities):        Formation of isoaspartyl peptide bonds is one of the most common        forms of non-enzymatic degradation of peptides and proteins        under mild conditions. For hGH, the primary site of succinimide        formation and subsequent hydrolysis to iso-aspartate and        aspartate is ASP130.    -   Degradation products (impurities) resulting from deamidation:        Under mild conditions, the primary deamidation sites of hGH are        ASN149 and ASN152.    -   Degradation products (impurities) resulting from oxidation:        Under mild conditions, the primary oxidation site of hGH is        MET14.

To enhance physicochemical or pharmacokinetic properties of a drug, suchas rhGH, in vivo, such drug can be conjugated with a carrier. If thedrug is transiently bound to a carrier and/or a linker, such systems arecommonly assigned as carrier-linked prodrugs. According to thedefinitions provided by IUPAC (as given underhttp://www.chem.qmul.ac.uk/iupac.medchem, accessed on Jul. 22, 2009), acarrier-linked prodrug is a prodrug that contains a temporary linkage ofa given active substance with a transient carrier group that producesimproved physicochemical or pharmacokinetic properties and that can beeasily removed in vivo, usually by a hydrolytic cleavage.

The linkers employed in such carrier-linked prodrugs are transient,meaning that they are non-enzymatically hydrolytically degradable(cleavable) under physiological conditions (aqueous buffer at pH 7.4,37° C.) with half-lives ranging from, for example, one hour to threemonths. The terms “transient” and “reversible” are used synonymously.

It is clear to the person skilled in the art that the hGH polymerprodrugs are covalent conjugates, meaning that the hGH moiety iscovalently attached to the polymer via the reversible linker moiety.

Preferably, the linkers employed in the hGH polymer prodrugs aretraceless, meaning that they release hGH in its free form.

Suitable carriers are polymers and can either be directly conjugated tothe linker or via a non-cleavable spacer. The term “hGH polymer prodrug”refers to carrier-linked prodrugs of hGH, wherein the carrier is apolymer.

The term polymer describes a molecule comprised of repeating structuralunits connected by chemical bonds in a linear, circular, branched,crosslinked or dendrimeric way or a combination thereof, which can be ofsynthetic or biological origin or a combination of both. Typically, apolymer has a molecular weight of at least 1 kDa.

Polymers are preferably selected from the group consisting of, forexample, 2-methacryloyl-oxyethyl phosphoyl cholins, hydrogels, PEG-basedhydrogels, poly(acrylic acids), poly(acrylates), poly(acrylamides),poly(alkyloxy) polymers, poly(amides), poly(amidoamines), poly(aminoacids), poly(anhydrides), poly(aspartamides), poly(butyric acids),poly(glycolic acids), polybutylene terephthalates, poly(caprolactones),poly(carbonates), poly(cyanoacrylates), poly(dimethylacrylamides),poly(esters), poly(ethylenes), poly(ethyleneglycols), poly(ethyleneoxides), poly(ethyl phosphates), poly(ethyloxazolines), poly(glycolicacids), poly(hydroxyethyl acrylates), poly(hydroxyethyloxazolines),poly(hydroxymethacrylates), poly(hydroxypropylmethacrylamides),poly(hydroxypropyl methacrylates), poly(hydroxypropyloxazolines),poly(iminocarbonates), poly(lactic acids), poly(lactic-co-glycolicacids), poly(methacrylamides), poly(methacrylates),poly(methyloxazolines), poly(organophosphazenes), poly(ortho esters),poly(oxazolines), poly(propylene glycols), poly(siloxanes),poly(urethanes), poly(vinyl alcohols), poly(vinyl amines),poly(vinylmethylethers), poly(vinylpyrrolidones), silicones, celluloses,carbomethyl celluloses, hydroxypropyl methylcelluloses, chitins,chitosans, dextrans, dextrins, gelatins, hyaluronic acids andderivatives, mannans, pectins, rhamnogalacturonans, starches,hydroxyalkyl starches, hydroxyethyl starches and othercarbohydrate-based polymers, xylans, and copolymers thereof.

The term “PEG” or “pegylation residue” is used herein exemplary forsuitable water-soluble polymers characterized by repeating units.Suitable polymers may be selected from the group consisting ofpolyalkyloxy polymers, hyaluronic acid and derivatives thereof,polyvinyl alcohols, polyoxazolines, polyanhydrides, poly(ortho esters),polycarbonates, polyurethanes, polyacrylic acids, polyacrylamides,polyacry-lates, polymethacrylates, polyorganophosphazenes,polysiloxanes, polyvinylpyrrolidone, polycyanoacrylates, and polyesters.Preferred are polyalkyloxy polymers, especially polyethylene glycolpolymers containing at least 10% by weight ethylene oxide units, morepreferably at least 25% by weight, even more preferably at least 50% byweight.

“Composition” refers to an intermixture of two or more chemicalsubstances. A pharmaceutical composition comprises the pharmaceuticallyactive moiety, either in its free form or as a prodrug, andpharmaceutically acceptable excipients and/or carriers.

“Dry composition” means that the hGH polymer prodrug composition isprovided in a dry form in a container. Suitable methods for drying arespray-drying and lyophilization (freeze-drying). Such dry composition ofhGH polymer prodrug has a residual water content of a maximum of 10%,preferably less than 5% and more preferably less than 2% (determinedaccording to Karl Fischer). The preferred method of drying islyophilization. “Lyophilized composition” means that the hGH polymerprodrug composition was first frozen and subsequently subjected to waterreduction by means of reduced pressure. This terminology does notexclude additional drying steps which occur in the manufacturing processprior to filling the composition into the final container.

“Lyophilization” (freeze-drying) is a dehydration process, characterizedby freezing a composition and then reducing the surrounding pressureand, optionally, adding heat to allow the frozen water in thecomposition to sublime directly from the solid phase to gas. Typically,the sublimed water is collected by desublimation.

“Reconstitution” means the addition of a liquid to bring back theoriginal form of a composition, such as a solution.

“Reconstitution solution” refers to the liquid used to reconstitute thedry composition of a rhGH polymer prodrug prior to administration to apatient in need thereof.

“Container” means any container in which the rhGH polymer prodrugcomposition is comprised and can be stored until reconstitution.

“Stable” and “stability” means that within the indicated storage timethe polymer conjugates remain conjugated and do not hydrolyze to asubstantial extent and exhibit an acceptable impurity profile relatingto rhGH. To be considered stable, the composition contains less than 5%of the drug in its free form and low amounts of rhGH-related impurities,such as ASP130 succinimide and isoaspartate formation, ASN140 and ASN152deamidation, and MET14 oxidation. Impurities may be quantified astryptic peptides based on their respective peak area relative to thepeak area of the corresponding unmodified tryptic peptide and “lowamounts” may correspond to an occurrence of such impurity to an extentof not greater than 20%, preferably not greater than 10%, even morepreferably not greater than 5% (per impurity).

“Free form” of a drug refers to the drug in its unmodified,pharmacologically fully active form, e.g. after being released from thepolymer conjugate.

“Therapeutically effective amount” means an amount sufficient to cure,alleviate or partially arrest the clinical manifestations of a givendisease and its complications. An amount adequate to accomplish this isdefined as “therapeutically effective amount”. Effective amounts foreach purpose will depend on the severity of the disease or injury aswell as the weight and general state of the subject. It will beunderstood that determining an appropriate dosage may be achieved usingroutine experimentation, by constructing a matrix of values and testingdifferent points in the matrix, which is all within the ordinary skillsof a trained physician. Within the scope of this invention,therapeutically effective amount relates to dosages that aim to achievetherapeutic effect for an extended period of time, i.e. for three daysor longer, for instance one week or two weeks.

“Buffer” or “buffering agent” refers to chemical compounds that maintainthe pH in a desired range. Physiologically tolerated buffers are, forexample, sodium phosphate, succinate, histidine, bicarbonate, citrateand acetate, sulphate, nitrate, chloride, pyruvate. Antacids such asMg(OH)₂ or ZnCO₃ may be also used. Buffering capacity may be adjusted tomatch the conditions most sensitive to pH stability.

“Excipients” refers to compounds administered together with thetherapeutic agent, for example, buffering agents, isotonicity modifiers,preservatives, stabilizers, anti-adsorption agents, oxidation protectionagents, or other auxiliary agents. However, in some cases, one excipientmay have dual or triple functions.

A “lyoprotectant” is a molecule which, when combined with a protein ofinterest, significantly prevents or reduces chemical and/or physicalinstability of the protein upon drying in general and especially duringlyophilization and subsequent storage. Exemplary lyoprotectants includesugars, such as sucrose or trehalose; amino acids such as monosodiumglutamate or histidine or arginine; methylamines such as betaine;lyotropic salts such as magnesium sulfate; polyols such as trihydric orhigher sugar alcohols, e.g. glycerin, erythritol, glycerol, arabitol,xylitol, sorbitol, and mannitol; ethylene glycol; propylene glycol;polyethylene glycol; pluronics; hydroxyalkyl starches, e.g. hydroxyethylstarch (HES), and combinations thereof.

Preferably, a “lyoprotectant” is a molecule which, when combined with aprotein of interest, significantly prevents or reduces chemical and/orphysical instability of the protein upon drying in general andespecially during lyophilization and subsequent storage. Exemplarylyoprotectants include sugars, such as sucrose or trehalose; amino acidssuch as monosodium glutamate or histidine; methylamines such as betaine;lyotropic salts such as magnesium sulfate; polyols such as trihydric orhigher sugar alcohols, e.g. glycerin, erythritol, glycerol, arabitol,xylitol, sorbitol, and mannitol; ethylene glycol; propylene glycol;polyethylene glycol; pluronics; hydroxyalkyl starches, e.g. hydroxyethylstarch (HES), and combinations thereof.

The lyoprotectant is preferably added in a “lyoprotecting amount” to thecomposition before the drying step, which means that, followinglyophilization of the protein in the presence of the lyoprotectingamount of the lyoprotectant, the protein essentially retains itsphysical and chemical stability and integrity upon lyophilization andstorage.

“Surfactant” refers to wetting agents that lower the surface tension ofa liquid.

“Isotonicity modifiers” refer to compounds which minimize pain that canresult from cell damage due to osmotic pressure differences at theinjection depot.

The term “stabilizers” refers to compounds used to stabilize the polymerprodrug. Stabilisation is achieved by strengthening of theprotein-stabilising forces, by destabilisation of the denatured state,or by direct binding of excipients to the protein.

“Anti-adsorption agents” refers to mainly ionic or non-ionic surfactantsor other proteins or soluble polymers used to coat or adsorbcompetitively to the inner surface of the composition's container.Chosen concentration and type of excipient depends on the effect to beavoided but typically a monolayer of surfactant is formed at theinterface just above the CMC value.

“Oxidation protection agents” refers to antioxidants such as ascorbicacid, ectoine, glutathione, methionine, monothioglycerol, morin,polyethylenimine (PEI), propyl gallate, vitamin E, chelating agents suchas citric acid, EDTA, hexaphosphate, thioglycolic acid.

“Antimicrobial” refers to a chemical substance that kills or inhibitsthe growth of microorganisms, such as bacteria, fungi, yeasts,protozoans and/or destroys viruses.

“Pharmaceutically acceptable” is meant to encompass any excipient and/oradditive, which does not interfere with the effectiveness of thebiological activity of the active ingredient and that, is not toxic tothe host to which it is administered.

“Sealing a container” means that the container is closed in such waythat it is airtight, allowing no gas exchange between the outside andthe inside and keeping the content sterile.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows exemplary structure 1 of type (AA1, AAA1), where the atleast 5 kDa polymer chain of S⁰ comprising G^(a) and BS¹ and BS² ismarked as S⁰; the carbamate group resulting from L^(a) and the primaryamino group of hGH is marked as L^(a); BS¹ comprises the at least 4 kDapolymer chain marked as S¹, wherein S¹ comprises BS³, which comprisesthe at least 4 kDa polymer chain marked as S³. BS² comprises the atleast 4 kDa polymer chain marked as S².

DETAILED DESCRIPTION OF EMBODIMENTS

It is to be understood that the figures and descriptions of the presentinvention have been simplified to illustrate elements that are relevantfor a clear understanding of the present invention, while eliminating,for purposes of clarity, many other elements which are conventional inthis art. Those of ordinary skill in the art will recognize that otherelements are desirable for implementing the present invention. However,because such elements are well known in the art, and because they do notfacilitate a better understanding of the present invention, a discussionof such elements is not provided herein.

The present invention will now be described in detail on the basis ofexemplary embodiments.

The compositions of the present invention contain rhGH polymer prodrugs.Preferably, the rhGH polymer prodrugs have the formula shown in (AB)hGH-(NH-L-S⁰)_(n)  (AB),wherein

-   n is 2, 3, or 4; preferably 2;-   hGH(—NH)_(n) represents the hGH residue;-   each L is a functional group L^(a), which is self hydrolysable    (auto-cleavable) by an auto-cleavage inducing group G^(a); and-   each S⁰ is independently a polymer chain having a molecular weight    of at least 5 kDa, wherein S⁰ is optionally branched by comprising    an at least first branching structure BS¹, the at least first    branching structure BS¹ comprising an at least second polymer chain    S¹ having a molecular weight of at least 4 kDa, wherein at least one    of S⁰, S¹ further comprises the auto-cleavage inducing group G^(a)    and wherein the molecular weight of the prodrug conjugate without    the hGH-NH is at least 25 kDa and at most 1000 kDa, preferably at    least 25 kDa and at most 500 kDa, even more preferably at least 30    kDa and at most 250 kDa, even more preferably at least 30 kDa and at    most 120 kDa, even more preferably at least 40 kDa and at most 100    kDa.

In a preferred embodiment, the polymer is PEG and the total PEG load pergrowth hormone molecule amounts to at least 25 kDa. Generally, the totalPEG load will be less than 1000 kDa. Preferably, the PEG load is atleast 25 kDa and at most 500 kDa, even more preferably at least 30 kDaand at most 250 kDa, even more preferably at least 30 kDa and at most120 kDa, even more preferably at least 40 kDa and at most 100 kDa, evenmore preferably at least 40 kDa and at most 90 kDa.

PEG may be attached to hGH through one or more anchoring points. In caseof one anchoring point, the corresponding PEG in the hGH PEG prodrugmonoconjugate will be branched and contains at least 3 chains. In caseof more than one anchoring point, such as in a bisconjugate, thecorresponding PEG in the hGH PEG prodrug may be branched or linear.Bisconjugates may contain linear or branched PEG or may contain amixture of one linear and one branched PEG chain. In case a branched PEGchain is used, there may be one or more branching units.

A branched PEG is a PEG molecule consisting of a branching pointconnecting two or more PEG chains, to form a molecule with one anchoringpoint for attachment to growth hormone. This could be two 20 kDa PEGchains joined to form one branched 40 kDa PEG molecule. In the casewhere the molecule contains two or three branching points, the moleculeis referred to 3 and 4 armed PEG, respectively.

In summary and within the restrictions mentioned above, the PEG polymeris not limited to a particular structure and can be linear, branched, ormulti-armed (e.g. forked PEG or PEG attached to a polyol core),dendritic, or with degradable linkers.

The PEGylation to native human GH may occur on several lysine groups oron the N-terminal amine (F1) as well described by Clark et al.(reference 2 herein) on page 21973 table III. Highly reactive arepositions F1 and LYS-140. Moderately reactive positions are LYS-115,LYS-38, and LYS-70. Poorly reactive are positions LYS-172, LYS-41,LYS-158 and LYS-168. However, PEGylation may occur at any lysineresidues of GH and/or at the N-terminal amine.

The rhGH polymer prodrugs of the dry compositions of the presentinvention are preferably PEGylated at one or more of the lysinesselected from the group consisting of Lys158, Lys145, Lys38, Lys140 andLys70. More preferably, PEGylation of the hGH moiety occurs mainly atpositions Lys158, Lys145, Lys38 and Lys140, even more preferably mainlyat positions Lys158, Lys145 and Lys38.

Preferably, at least 30% of all growth hormone moieties of thecomposition of the present invention are PEGylated at position Lys158.

In this context, the phrase “PEGylation occurs mainly at position LysX”means that at least 10% of all rhGH moieties of the rhGH polymerprodrugs are PEGylated at amino acid position X.

In more general terms the PEG used herein in combination with atransient linker may reduce the risk of lipoatrophy by suitable choiceof said polymer. However the principles of the present invention alsoapply to polymers other than PEG. Thus the term PEG is only used hereinexemplary for suitable polymers.

Thus, in a preferred embodiment, hGH PEG prodrug is a monoconjugateconjugated with one of its primary amino groups to an auto-cleavablefunctional group L^(a) to a polymer chain S⁰. This polymer chain S⁰ hasa molecular weight of at least 5 kDa and comprises at least onebranching structure BS¹. The branching structure BS¹ comprises a secondpolymer chain S¹, which has a molecular weight of at least 4 kDa.

As outlined above, at least a third polymer chain S² is required to havea molecular weight of at least 4 kDa. The polymer chain S² may be a partof BS¹ or may be a further branch of S⁰ or S¹ resulting in a furtherbranching structure BS², which comprises S².

Optionally, more than 3 polymer chains are present in the prodrugconjugate comprised in the dry compositions of the present invention,e.g. 4, 5, 6, 7, or 8. However each further polymer chain has amolecular weight of at least 4 kDa. The total number of polymer chainsis limited by the total weight of the prodrug conjugate being at most1000 Da (without hGH-NH).

Thus, a preferred embodiment of the rhGH polymer prodrug comprised inthe dry composition of the present invention relates to prodrug, whereinat least one of the branching structures BS¹, BS² comprises a furtherfourth polymer chain S³ having a molecular weight of at least 4 kDa orone of S⁰, S¹, S² comprises a third branching structure BS³ comprisingthe at least fourth polymer chain S³ having a molecular weight of atleast 4 kDa.

The auto-cleavage inducing group G^(a), which is necessary for theauto-cleavage of L^(a) is comprised by one of the branching structuresor polymer chains.

Optionally, one of the branching structures serves as group G^(a) sothat the branching structure consists of G^(a) (instead of comprisingsaid group), which is also encompassed by the term “comprising”.

FIG. 1 illustrates the meaning of L^(a), G^(a), S⁰, S¹, S², S³, BS¹, BS²and BS³ using an exemplary hGH polymer prodrug.

The preparation of a prodrug conjugate (AA) normally results in amixture of conjugates, where several primary amino groups of hGH areconjugated resulting in different mono-conjugated, differentbi-conjugated, different tri-conjugated, etc., prodrugs. Correspondingmonoconjugated, bisconjugated or trisconjugated hGH PEG prodrugs can beseparated by standard methods known in the art, like columnchromatography and the like.

In monoconjugates of hGH PEG prodrugs, the at least three polymer chainsS¹, S² contain a “polymer moiety”, which is characterized by one or morerepeating units, which may be randomly, block wise or alternatingdistributed. In addition, the at least three polymer chains S⁰, S¹, S²show an end group, which is typically a hydrogen atom or an alkyl grouphaving from 1 to 6 carbon atoms, which may be branched or unbranched,e.g. a methyl group, especially for PEG based polymer chains resultingin so called mPEGs. The term “PEG-based” as understood herein is appliedto a polymer of which the mass proportion of PEG is at least 10% byweight, preferably at least 25%, and more preferably at least 50% basedon the total weight of the polymer chain.

It is pointed out that the polymer moieties within the at least threepolymer chains S⁰, S¹, S² may have further chain-like substituents,originating from the repeating units and resulting in chains having lessthan 4 kDa of molecular weight and which are not considered as polymerchains S⁰, S¹, S², etc. Preferably, the at least three polymer chainsS⁰, S¹, S² carry substituents of less than 1000 Da molecular weight.

A relevant structural feature of S⁰ is its critical distance. Thecritical distance defines the shortest distance between the attachmentsite of S⁰ to L^(a) and the first branching structure BS¹ measured asconnected atoms. The length of the critical distance has an effect onthe residual activity as discussed for compound 33 as described in WO-A2009/133137, which is hereby incorporated by reference. The criticaldistance is preferably less than 50, more preferred less than 20, andmost preferred less than 10.

The at least three polymer chains S⁰, S¹ and S² typically each containan interconnecting moiety. Ga is present in at least one of theinterconnecting moieties. For polymer chains other than S⁰, theinterconnecting moiety is the structural element connecting the polymermoiety of for instance S¹ with BS¹ and the polymer moiety of S² withBS². For S⁰, the interconnecting moiety is the structural elementconnecting L^(a) and BS¹.

Interconnecting moieties may consist of a C₁₋₅₀ alkyl chain, which isbranched or unbranched and which is optionally interrupted or terminatedby hetero atoms or functional groups selected from the group consistingof —O—; —S—; N(R); C(O); C(O)N(R); N(R)C(O); one or more carbocycles orheterocycles, wherein R is hydrogen or a C₁₋₂₀ alkyl chain, which isoptionally interrupted or terminated by one or more of theabovementioned atoms or groups, which further have a hydrogen asterminal atom; and wherein a carbocycle is phenyl; naphthyl; indenyl;indanyl; tetralinyl; C₃₋₁₀ cycloalkyl; and wherein the heterocycle is a4 to 7 membered heterocyclyl; or 9 to 11 membered heterobicyclyl.

“C₃₋₁₀ cycloalkyl” or “C₃₋₁₀ cycloalkyl ring” means a cyclic alkyl chainhaving 3 to 10 carbon atoms, which may have carbon-carbon double bondsbeing at least partially saturated, e.g. cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl,cyclononyl, cyclodecyl. Each hydrogen of a cycloalkyl carbon may bereplaced by a substituent. The term “C₃₋₁₀ cycloalkyl” or “C₃₋₁₀cycloalkyl ring” also includes bridged bicycles like norbonane ornorbonene.

“4 to 7 membered heterocyclyl” or “4 to 7 membered heterocycle” means aring with 4, 5, 6 or 7 ring atoms that may contain up to the maximumnumber of double bonds (aromatic or non-aromatic ring which is fully,partially or un-saturated) wherein at least one ring atom up to 4 ringatoms are replaced by a heteroatom selected from the group consisting ofsulfur (including —S(O)—, —S(O)₂—), oxygen and nitrogen (including═N(O)—) and wherein the ring is linked to the rest of the molecule via acarbon or nitrogen atom. Examples for a 4 to 7 membered heterocycles areazetidine, oxetane, thietane, furan, thiophene, pyrrole, pyrroline,imidazole, imidazoline, pyrazole, pyrazoline, oxazole, oxazoline,isoxazole, is oxazoline, thiazole, thiazoline, isothiazole,isothiazoline, thiadiazole, thiadiazoline, tetrahydrofuran,tetrahydrothiophene, pyrrolidine, imidazolidine, pyrazolidine,oxazolidine, is oxazolidine, thiazolidine, is othiazolidine,thiadiazolidine, sulfolane, pyran, dihydropyran, tetrahydropyran,imidazolidine, pyridine, pyridazine, pyrazine, pyrimidine, piperazine,piperidine, morpholine, tetrazole, triazole, triazolidine,tetrazolidine, diazepane, azepine or homopiperazine.

“9 to 11 membered heterobicyclyl” or “9 to 11 membered heterobicycle”means a heterocyclic system of two rings with 9 to 11 ring atoms, whereat least one ring atom is shared by both rings and that may contain upto the maximum number of double bonds (aromatic or non-aromatic ringwhich is fully, partially or un-saturated) wherein at least one ringatom up to 6 ring atoms are replaced by a heteroatom selected from thegroup consisting of sulfur (including —S(O)—, —S(O)₂—), oxygen andnitrogen (including ═N(O)—) and wherein the ring is linked to the restof the molecule via a carbon or nitrogen atom. Examples for a 9 to 11membered heterobicycle are indole, indoline, benzofuran, benzothiophene,benzoxazole, benzisoxazole, benzothiazole, benzisothiazole,benzimidazole, benzimidazoline, quinoline, quinazoline,dihydroquinazoline, quinoline, dihydroquinoline, tetrahydroquinoline,decahydroquinoline, isoquinoline, decahydroisoquinoline,tetrahydroisoquinoline, dihydroisoquinoline, benzazepine, purine orpteridine. The term 9 to 11 membered heterobicycle also includes spirostructures of two rings like 1,4-dioxa-8-azaspiro[4.5]decane or bridgedheterocycles like 8-aza-bicyclo[3.2.1]octane.

The carbocycle, heterocycle and heterobicycle may be substituted byC₁₋₂₀ alkyl, optionally interrupted or terminated by hetero atoms orfunctional groups selected from the group consisting of —O—; —S—; N(R);C(O); C(O)N(R); N(R)C(O), wherein R is hydrogen or a C₁₋₁₀ alkyl chain,which is optionally interrupted or terminated by one or more of theabovementioned atoms or groups, which further have a hydrogen asterminal atom.

The polymer moiety of the at least three chains S⁰, S¹, S² form themajority part of the chains, preferably at least 90% of the molecularweight of each chain, more preferred at least 95%, even more preferredat least 97.5%, even more preferred at least 99%. Thus, the basis of thechains is represented by the polymer moiety.

Preferably, the at least three chains S⁰, S¹, S² are independently basedon a polymer selected from the group consisting of polyalkyloxypolymers, hyaluronic acid and derivatives thereof, polyvinyl alcohols,polyoxazolines, polyanhydrides, poly(ortho esters), polycarbonates,polyurethanes, polyacrylic acids, polyacrylamides, polyacrylates,polymethacrylates, polyorganophosphazenes, polysiloxanes,polyvinylpyrrolidone, polycyanoacrylates, and polyesters.

Preferably, the at least three chains S⁰, S¹, S² are based on the samepolymer. Preferably, the at least three chains S⁰, S¹, S² are based onpolyalkyoxy polymers. Even more preferred the at least three chains S⁰,S¹, S² are polyethylene glycol based.

The same applies for further chains S³, S⁴, S⁵, etc, accordingly.

The chain S⁰ comprises a branching structure BS¹, so that S¹ is linkedto S⁰. For the linkage of S² the branching structure BS¹ may be used ora further branching structure BS² is present, which may be a part S⁰ orS¹. Accordingly, further branching structures may be present, whenfurther chains are present. For example in case a chain S³ is present itmay be linked to BS¹, BS² or a branching structure BS³. The branchingstructure BS³, if present, may be part of S⁰, S¹, or S².

In general any chemical entity, which allows the branching of a chain,may be used. Preferably, the branching structures are independentlyselected from the group consisting of at least 3-fold substitutedcarbocycle, at least 3-fold substituted heterocycle, a tertiary carbonatom, a quaternary carbon atom, and a tertiary nitrogen atom, whereinthe terms carbocycle and heterocycle are defined as indicated above.

In publications in the field of auto-cleavage inducing groups aresometimes called linkers to discriminate their structure from thecarrier. Nevertheless it is often difficult to clearly separate thesestructural features. Therefore, within the meaning of the presentinvention the cleavage-inducing group G^(a) is considered to be part ofthe carrier S, comprising at least S⁰, S¹, S², BS¹, and optionally BS².Variation of the chemical nature of G^(a) allows the engineering of theproperties of the self-cleaving properties of a correspondingcarrier-linked prodrug to a great extent.

As discussed above, a PEGylated-prodrug, wherein the drug is for examplerhGH as described in patent application WO-A 2005/099768, and has acharacteristic of release, which is therein described as the 1,6cleavage system without the production of toxic aromatic compounds. Inthis document is broadly described numerous herein relevant suitabletransient linker structures to get a relevant release profile ofinterest. Other transient linker structures are generically/broadlydescribed in e.g. other Complex Biosystems GmbH applications such asWO-A 2005/034909, WO-A 2005/099768, WO-A 2006/003014 and WO-A2006/136586.

More transient linker structures are broadly described in e.g. WO-A99/30727 (Enzon Inc).

In order to solve the present problems for GH as discussed herein, thepresent inventors have selected suitable preferred transient linkerstructures to get the herein described relevant functional properties ofthe rhGH PEGylated prodrug. Based on the herein detailed description ofpreferred linker structures it is within the skilled person knowledge tomake other suitable preferred transient linker structures that couldgive an rhGH PEGylated prodrug with the herein described relevantfunctional properties.

Especially, suitable transient linker structures, which are selfhydrolysable (auto-cleavable) can be chosen for incorporation into S⁰.The herein selected linker structures are described in detail below.

In order to introduce hydrolytic lability into functional groups L suchas amides or carbamates, it is necessary to engineer structural chemicalcomponents into the carrier in order to function, for instance, asneighbouring groups in proximity to the functional group. Suchautocleavage-inducing chemical structures that exert control over thecleavability of the prodrug amide bond are termed auto-cleavage inducinggroups G^(a). Autocleavage-inducing groups can have a strong effect onthe rate of hydrolysis of a given functional group L^(a).

Preferred L^(a) are selected from the group consisting of C(O)—O—, andC(O)—, which form together with the primary amino group of hGH acarbamate or amide group.

Thus, a composition of the present invention is preferred, wherein L^(a)is selected from the group consisting of C(O)—O—, and C(O)—, which formtogether with the primary amino group of hGH a carbamate or amide groupresulting in formula (AA1) or (AA2)hGH-NH—C(O)O—S⁰  (AA1),hGH-NH—C(O)—S⁰  (AA2).

The following sections will list various structural components that mayfunction as cleavage-inducing groups G^(a).

The group G^(a) represents an autocleavage inducing group. G^(a) may bepresent as such or as a cascade autocleavage-inducing group, which isunmasked to become effective by means of an additional hydrolytical orenzymatic cleavage step. If G^(a) is present as such, it governs therate-limiting autohydrolysis of L^(a).

Examples for G^(A):

A. J. Garman et al. (A. J. Garman, S. B. Kalindjan, FEBS Lett. 1987, 223(2), 361-365 1987) used PEG5000-maleic anhydride for the reversiblemodification of amino groups in tissue-type plasminogen activator andurokinase. Regeneration of functional enzyme from PEG-uPA conjugate uponincubation at pH 7.4 buffer by cleavage of the maleamic acid linkeagefollowed first order kinetics with a half-life of 6.1 h.

Simple aromatic moieties may infer lability to a connected carbamatebond (WO-A 01/47562). For instance, substituted or unsubstitutedfluorenylmethyl group were used to labilize carbamate linkages tovarious bioactive agents in a prodrug approach (Tsubery et al. J BiolChem 279 (2004) 38118-24). Two PEG chains were attached to a fluorenylmoiety in WO-A 2007/075534.

Thus, G^(a) is an aromatic ring or fluorenylmethyl directly attached toa carbamate functional group L^(a).

Accordingly, a composition of the present invention is preferred,wherein G^(a) is an aromatic ring or fluorenylmethyl directly attachedto a carbamate functional group formed by L^(a) and the primary aminogroup of hGH.

Alternatively, transformation of G^(a) may induce a molecularrearrangement within S⁰ such as a 1,4- or 1,6-elimination. Therearrangement renders L^(a) more labile so that its cleavage is induced.The transformation of G^(a) is the rate-limiting step in the cascademechanism. Ideally, the cleavage rate of the temporary linkage isidentical to the desired release rate for the drug molecule in a giventherapeutic scenario. In such a cascade system based on 1,6-elimination,it is desirable that the cleavage of L^(a) is substantiallyinstantaneous after its lability has been induced by transformation ofG^(a). In addition it is desirable that the rate-limiting cleavagekinetics proceed in a therapeutically useful timeframe without therequirement for additional enzymatic contribution in order to avoid thedrawbacks associated with predominantly enzymatic cleavage discussedabove.

R. B. Greenwald, A. Pendri, C. D. Conover, H. Zhao, Y. H. Choe, A.Martinez, K. Shum, S. Guan, J. Med. Chem., 1999, 42, 3657-3667 & PCTPatent Application WO-A 99/30727 described a methodology forsynthesizing poly(ethylene glycol) prodrugs of amino-containing smallmolecule compounds based on 1,4- or 1,6-benzyl elimination. In thisapproach the amino group of the drug molecule is linked via a carbamategroup to a PEGylated benzyl moiety. The poly(ethylene glycol) wasattached to the benzyl group by ester, carbonate, carbamate, or amidebonds. The release of PEG from the drug molecule occurs through acombination of autohydrolysis and enzymatic cleavage. The cleavage ofthe release-triggering masking group is followed in this approach by theclassical and rapid 1,4- or 1,6-benzyl elimination. This linker systemwas also used for releasable poly(ethylene glycol) conjugates ofproteins (S. Lee, R. B. Greenwald et al. Bioconj. Chem. 2001, 12 (2),163-169). Lysozyme was used as model protein because it loses itsactivity when PEGylation takes place on the epsilon-amino group oflysine residues. Various amounts of PEG linker were conjugated to theprotein. Regeneration of native protein from the PEG conjugates occurredin rat plasma or in non-physiological high pH buffer. See also F. M. H.DeGroot et al. (WO-A 2002/083180 and WO-A 2004/043493), and D. Shabat etal. (WO-A 2004/019993).

Thus, L^(a) is a carbamate functional group, the cleavage of said groupis induced by a hydroxyl or amino group of G^(a) via 1,4- or 1,6 benzylelimination of S⁰, wherein G^(a) contains ester, carbonate, carbamate,or amide bonds that undergo rate-limiting transformation. In effect,G^(a) may be cleaved off by hydrolysis.

Accordingly, a composition of the present invention is preferred,wherein L^(a) forms together with the amino group of hGH a carbamatefunctional group, the cleavage of said group is induced by a hydroxyl oramino group of G^(a) via 1,4- or 1,6 benzyl elimination of S⁰, whereinG^(a) contains ester, carbonate, carbamate, or amide bonds that undergorate-limiting transformation.

G^(a) may contain a cascade cleavage system that is enabled bycomponents of G^(a) that are composed of a structural combinationrepresenting the aforementioned precursor. A precursor of G^(a) maycontain additional temporary linkages such as an amide, ester or acarbamate. The stability or susceptibility to hydrolysis of theprecursor's temporary linkage (e.g. carbamate) may be governed byautohydrolytic properties or may require the activity of an enzyme.

Antczak et al. (Bioorg Med Chem 9 (2001) 2843-48) describe a reagentwhich forms the basis for a macromolecular cascade prodrug system foramine-containing drug molecules. In this approach an antibody serves asthe carrier, a stable bond connects the antibody to an activating group,carrying a cleavable masking group. Upon removal of the ester-linkedmasking group, L^(a) cleaves and releases the drug compound.

D. Shabat et al. (Chem. Eur. 3. 2004, 10, 2626-2634) describe a polymerprodrug system based on a mandelic acid activating group. In this systemthe masking group is linked to the activating group by a carbamate bond.The activating group is conjugated permanently to a polyacrylamidepolymer via an amide bond. After activation of the masking group by acatalytic antibody, the masking group is cleaved by cyclization and thedrug is released. The activating group is still connected to thepolyacrylamide polymer after drug release.

M.-R. Lee et al. describe (Angew. Chem. 2004, 116, 1707-17 10) a similarprodrug system based on a mandelic acid activating group and anester-linked masking group. Nevertheless in these linkers a1,6-elimination step still generates a highly reactive aromaticintermediate. Even if the aromatic moiety remains permanently attachedto the polymer carrier, side reactions with potentially toxic orimmunogenic effects may be caused.

Greenwald et al. published in 2000 a poly(ethylene glycol) drug deliverysystem of amino-containing prodrugs based on trimethyl locklactonization (R. B. Greenwald et al. J. Med. Chem. 2000, 43(3),457-487; WO-A 02/089789). In this prodrug system substitutedo-hydroxyphenyl-dimethylpropionic acid is coupled to amino groups ofdrug molecules by an amide bond. The hydroxy group is linked to PEG byan ester, carbonate, or carbamate group. The rate determining step indrug release is the enzymatic cleavage of these functional groupsfollowed by fast amide cleavage by lactonization, liberating an aromaticlactone side product.

More recently, R. B. Greenwald et al. (Greenwald et al. J. Med. Chem.2004, 47, 726-734) described a PEG prodrug system based onbis-(N-2-hydroxyethyl)glycin amide (bicin amide) linker. In this systemtwo PEG molecules are linked to a bicin molecule coupled to an aminogroup of the drug molecule. The first two steps in prodrug activation isthe enzymatic cleavage of both PEG molecules. Different linkages betweenPEG and bicin are described resulting in different prodrug activationkinetics. The main disadvantage of this system is the slow hydrolysisrate of bicin amide conjugated to the drug molecule (t_(1/2)=3 h inphosphate buffer) resulting in the release of a bicin-modified prodrugintermediate that may show different pharmacokinetic and pharmacodynamicproperties than the parent drug molecule.

More specifically, preferred groups L^(a) and G^(a) with specific spacermoieties for S⁰ are described below.

A preferred structure according to WO-A 2005/099768 is selected from thegeneral formula (I) and (II):

-   wherein in these formulae T represents hGH-NH; X represents a spacer    moiety; Y₁ and Y₂ each independently represent O, S or NR₆; Y₃ and    Y₅ independently of each other represent O or S; Y₄ represents O,    NR₆ or —C(R7)(R8); R2 and R3 independently of each other represent a    moiety selected from the group consisting of hydrogen, substituted    or unsubstituted linear, branched or cyclical alkyl or heteroalkyl    groups, aryls, substituted aryls, substituted or unsubstituted    heteroaryls, cyano groups, nitro groups, halogens, carboxy groups,    carboxyalkyl groups, alkylcarbonyl groups or carboxamidoalkyl    groups; R4 represents a moiety selected from the group consisting of    hydrogen, substituted or unsubstituted linear, branched or cyclical    alkyls or heteroalkyls, aryls, substituted aryls, substituted or    unsubstituted heteroaryl, substituted or unsubstituted linear,    branched or cyclical alkoxys, substituted or unsubstituted linear,    branched or cyclical heteroalkyloxys, aryloxys or heteroaryloxys,    cyano groups and halogens; R7 and R8 are each independently selected    from the group consisting of hydrogen, substituted or unsubstituted    linear, branched or cyclical alkyls or heteroalkyls, aryls,    substituted aryls, substituted or unsubstituted heteroaryls,    carboxyalkyl groups, alkylcarbonyl groups, carboxamidoalkyl groups,    cyano groups, and halogens; R6 represents a group selected from    hydrogen, substituted or unsubstituted linear, branched or cyclical    alkyls or heteroalkyls, aryls, substituted aryls and substituted or    unsubstituted heteroaryls; R1 represents the rest of S⁰; W    represents a group selected from substituted or unsubstituted    linear, branched or cyclical alkyls, aryls, substituted aryls,    substituted or unsubstituted linear, branched or cyclical    heteroalkyls, substituted or unsubstituted heteroaryls; Nu    represents a nucleophile; n represents zero or a positive imager;    and Ar represents a multi-substituted aromatic hydrocarbon or    multi-substituted aromatic heterocycle.

Within the meaning of the present invention, the group L^(a) isrepresented by Y₃—C(Y₅)NH— (together with the amino group of hGH), G^(a)is represented by Nu-W—Y₄—C(Y₁)Y₂ and Ar(R4)_(n)—C(R3)XR₁ represents S⁰,which further includes at least S¹, S², BS¹ and optionally BS².

In an alternative embodiment S¹ is attached via Ar or represents R3.Then the carbon atom adjacent to Y₃ substituted with XR1 represents thebranching structure BS¹, S¹ is terminated with Ar comprising G^(a). itis evident that in this embodiment terms S⁰ and S¹ are interchangeable.

Preferably, in formula (AA) or (AA1) S⁰ is of formula (AAA1)

-   wherein in formula (AAA1)-   G^(a) has the meaning as indicated above;-   S⁰⁰ is CH₂; or C(O);-   S^(OA) is an alkylene chain having from 1 to 20 carbon atoms, which    is optionally interrupted or terminated by one or more groups,    cycles or heteroatoms selected from the group consisting of    optionally substituted heterocycle; O; S; C(O); and NH;-   BS¹, BS², BS³ are independently selected from the group consisting    of N; and CH.-   S^(0B), S^(1A) are independently an alkylene chain having from 1 to    200 carbon atoms, which is optionally interrupted or terminated by    one or more groups, cycles or heteroatoms selected from the group    consisting of optionally substituted heterocycle; O; S; C(O); and    NH;-   S^(0C), S^(1B), are (C(O))_(n2)(CH₂)_(n1)(OCH₂CH₂)_(n)OCH₃, wherein    each n is independently an integer from 100 to 500, each n1 is    independently 0, 1, 2, 3, 4, 5, 6, 7, or 8, and n2 is 0 or 1.-   S², S³ are independently hydrogen; or    (C(O))_(n2)(CH₂)_(n1)(OCH₂CH₂)_(n)OCH₃, wherein each n is    independently an integer from 100 to 500, each n1 is independently    0, 1, 2, 3, 4, 5, 6, 7, or 8, and n2 is 0 or 1, provided that at    least one of S², S³ is other than hydrogen;-   R², R³ are defined as for formula (A) below.

The term heterocycle means a heterocycle as defined above. Optionalsubstituents are, e.g. oxo (═O), where the ring is at least partiallysaturated, a branched or unbranched alkyl chain having from one to 6carbon atoms, or halogen. A preferred substituted heterocycle issuccinimide.

Preferably, G^(a) in formula (AAA1) is OC(O)—R and R is the partialstructure of formula (I) as shown below, wherein R1, R4, R5 and n aredefined as given below.

Another preferred embodiment is described in WO06136586A2. Accordingly,the following structures are preferred:

-   wherein in these three structures T is NH-hGH;-   X is a spacer moiety such as R13-Y1;-   Y1 is O, S, NR6, succinimide, maleimide, unsaturated carbon-carbon    bonds or any heteratom containing a free electron pair or is absent;-   R13 is selected from substituted or non-substituted linear, branched    or cyclical alkyl or heteroalkyl, aryls, substituted aryls,    substituted or non-substituted heteroaryls;-   R2 and R3 are selected independently from hydrogen, acyl groups, or    protecting groups for hydroxyl groups;-   R4 to R12 are selected independently from hydrogen, X—R1,    substituted or non-substituted linear, branched or cyclical alkyl or    heteroalkyl, aryls, substituted aryls, substituted or    non-substituted heteroaryls, cyano, nitro, halogen, carboxy,    carboxamide;-   R1 is the rest of S⁰, comprising at least S¹, S², BS¹, and    optionally BS².

In this embodiment L^(a) is an amide group, and G^(a) encompasses theN-branched structure carrying OR₂/OR₃.

In yet another preferred embodiment, a preferred structure is given by aprodrug conjugate D-L, wherein

-   -   D is NH-hGH; and    -   L is a non-biologically active linker moiety -L′ represented by        formula (I),

-   wherein in formula (I) the dashed line indicates the attachment to    the amino group of hGH by forming an amide bond;-   X is C(R⁴R^(4a)); N(R⁴); O; C(R⁴R^(4a))—C(R⁵R^(5a));    C(R⁵R^(5a))—C(R⁴R^(4a)); C(R⁴R^(4a))—N(R⁶); N(R⁶)—C(R⁴R^(4a));    C(R⁴R^(4a))—O; or O—C(R⁴R^(4a));-   X¹ is C; or S(O);-   X² is C(R⁷, R^(7a)); or C(R⁷, R^(7a))—C(R⁸, R^(8a));-   R¹, R^(1a), R², R^(2a), R³, R^(3a), R⁴, R^(4a), R⁵, R^(5a), R⁶, R⁷,    R^(7a), R⁸, R^(8a) are independently selected from the group    consisting of H; and C₁₋₄ alkyl; or-   Optionally, one or more of the pairs R^(1a)/R^(4a), R^(1a)/R^(5a),    R^(4a)/R^(5a), R^(4a)/R^(5a), R^(7a)/R^(8a) form a chemical bond;-   Optionally, one or more of the pairs R^(1a)/R^(1a), R^(2a)/R^(2a),    R^(4a)/R^(4a), R^(5a)/R^(5a), R⁷/R^(7a), R⁸/R^(8a) are joined    together with the atom to which they are attached to form a C₃₋₇    cycloalkyl; or 4 to 7 membered heterocyclyl;-   Optionally, one or more of the pairs R¹/R⁴, R¹/R⁵, R¹/R⁶, R⁴/R⁵,    R⁷/R⁸, R²/R³ are joined together with the atoms to which they are    attached to form a ring A;-   Optionally, R³/R^(3a) are joined together with the nitrogen atom to    which they are attached to form a 4 to 7 membered heterocycle;-   A is selected from the group consisting of phenyl; naphthyl;    indenyl; indanyl; tetralinyl; C₃₋₁₀ cycloalkyl; 4 to 7 membered    heterocyclyl; and 9 to 11 membered heterobicyclyl; and-   wherein L¹ is substituted with one group L²-Z and optionally further    substituted, provided that the hydrogen marked with the asterisk in    formula (I) is not replaced by a substituent; wherein-   L² is a single chemical bond or a spacer; and-   Z is the rest of S⁰, comprising at least S¹, S², BS¹, and optionally    BS².

In this embodiment L^(a) is represented by an amide group and G^(a) isrepresented by N(H*)X¹(O) and the chain connecting to N includingsubstituents of N.

Prodrug conjugates of this type are described in European Patentapplication No 08150973.9

Accordingly, a composition of the present invention is preferred,wherein L^(a)-S⁰ is represented by formula (AAA2),

-   wherein in formula (AAA2) the dashed line indicates the attachment    to the primary amino group of hGH so that L^(a) and the amino group    form an amide bond;-   X is C(R⁴R^(4a)); N(R⁴); O; C(R⁴R^(4a))—C(R⁵R^(5a));    C(R⁵R^(5a))—C(R⁴R^(4a)); C(R⁴R^(4a))—N(R⁶); N(R⁶)—C(R⁴R^(4a));    C(R⁴R^(4a))—O; or O—C(R⁴R^(4a));-   X¹ is C; or S(O);-   X² is C(R⁷, R^(7a)); or C(R⁷, R^(7a))—C(R⁸, R^(8a));-   R¹, R^(1a), R², R^(2a), R³, R^(3a), R⁴, R^(4a), R⁵, R^(5a), R⁶, R⁷,    R^(7a), R⁸, R^(8a) are independently selected from the group    consisting of H; and C₁₋₄ alkyl; or

Optionally, one or more of the pairs R^(1a)/R^(4a), R^(1a)/R^(5a),R^(4a)/R^(5a), R^(4a)/R^(5a), R^(7a)/R^(8a) form a chemical bond;

Optionally, one or more of the pairs R^(1a)/R^(1a), R^(2a)/R^(2a),R^(4a)/R^(4a), R^(5a)/R^(5a), R⁷/R^(7a), R⁸/R^(8a) are joined togetherwith the atom to which they are attached to form a C₃₋₇ cycloalkyl; or 4to 7 membered heterocyclyl;

-   Optionally, one or more of the pairs R¹/R⁴, R¹/R⁵, R¹/R⁶, R⁴/R⁵,    R⁷/R⁸, R²/R³ are joined together with the atoms to which they are    attached to form a ring A;-   Optionally, R³/R^(3a) are joined together with the nitrogen atom to    which they are attached to form a 4 to 7 membered heterocycle;-   A is selected from the group consisting of phenyl; naphthyl;    indenyl; indanyl; tetralinyl; C₃₋₁₀ cycloalkyl; 4 to 7 membered    heterocyclyl; and 9 to 11 membered heterobicyclyl; and-   wherein S⁰ is substituted with one group L²-Z and optionally further    substituted, provided that the hydrogen marked with the asterisk in    formula (AAA2) is not replaced by a substituent; wherein-   L² is a single chemical bond or a spacer; and-   Z is of formula (AAA2a)

-   -   wherein S⁰⁰, S^(0A), S^(0B), S^(0C), S^(1A), S^(1B), S², S³,        BS¹, BS², and BS³ have the meaning as indicated for formula        (AAA1) above.

“Alkyl” means a straight-chain or branched carbon chain. Each hydrogenof an alkyl carbon may be replaced by a substituent.

“C₁₋₄ alkyl” means an alkyl chain having 1-4 carbon atoms, e.g. ifpresent at the end of a molecule: methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, sec-butyl tert-butyl, or e.g. —CH₂—, —CH₂—CH₂—,—CH(CH₃)—, —CH₂—CH₂—CH₂—, —CH(C₂H₅)—, —C(CH₃)₂—, when two moieties of amolecule are linked by the alkyl group. Each hydrogen of a C₁₋₄ alkylcarbon may be replaced by a substituent.

“C₁₋₆ alkyl” means an alkyl chain having 1-6 carbon atoms, e.g. ifpresent at the end of a molecule: C₁₋₄ alkyl, methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl; tert-butyl, n-pentyl, n-hexyl,or e.g. —CH₂—, —CH₂—CH₂—, —CH(CH₃)—, —CH₂—CH₂—CH₂—, —CH(C₂H₅)—,—C(CH₃)₂—, when two moieties of a molecule are linked by the alkylgroup. Each hydrogen of a C₁₋₆ alkyl carbon may be replaced by asubstituent. The term “C₁₋₈ alkyl” is defined accordingly.

Accordingly, “C₁₋₁₈ alkyl” means an alkyl chain having 1 to 18 carbonatoms and “C₈₋₁₈ alkyl” means an alkyl chain having 8 to 18 carbonatoms. Accordingly, “C₁₋₅₀ alkyl” means an alkyl chain having 1 to 50carbon atoms.

“C₂₋₅₀ alkenyl” means a branched or unbranched alkenyl chain having 2 to50 carbon atoms, e.g. if present at the end of a molecule: —CH═CH₂,—CH═CH—CH₃, —CH₂—CH═CH₂, —CH═CH—CH₂—CH₃, —CH═CH—CH═CH₂, or e.g. —CH═CH—,when two moieties of a molecule are linked by the alkenyl group. Eachhydrogen of a C₂₋₅₀ alkenyl carbon may be replaced by a substituent asfurther specified. Accordingly, the term “alkenyl” relates to a carbonchain with at least one carbon carbon double bond. Optionally, one ormore triple bonds may occur.

“C₂₋₅₀ alkynyl” means a branched or unbranched alkynyl chain having 2 to50 carbon atoms, e.g. if present at the end of a molecule: —C≡CH,—CH₂—C≡CH, CH₂—CH₂—C≡CH, CH₂—C≡C—CH₃, or e.g. —C≡C— when two moieties ofa molecule are linked by the alkynyl group. Each hydrogen of a C₂₋₅₀alkynyl carbon may be replaced by a substituent as further specified.Accordingly, the term “alkynyl” relates to a carbon chain with at leastone carbon carbon triple bond. Optionally, one or more double bonds mayoccur.

“C₃₋₇ cycloalkyl” or “C₃₋₇ cycloalkyl ring” means a cyclic alkyl chainhaving 3 to 7 carbon atoms, which may have carbon-carbon double bondsbeing at least partially saturated, e.g. cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl. Each hydrogen of acycloalkyl carbon may be replaced by a substituent. The term “C₃₋₇cycloalkyl” or “C₃₋₇ cycloalkyl ring” also includes bridged bicycleslike norbonane or norbonene. Accordingly, “C₃₋₅ cycloalkyl” means acycloalkyl having 3 to 5 carbon atoms.

Accordingly, “C₃₋₁₀ cycloalkyl” means a cyclic alkyl having 3 to 10carbon atoms, e.g. C₃₋₇ cycloalkyl; cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl,cyclononyl, cyclodecyl. The term “C₃₋₁₀ cycloalkyl” also includes atleast partially saturated carbomono- and -bicycles.

“Halogen” means fluoro, chloro, bromo or iodo. It is generally preferredthat halogen is fluoro or chloro.

“4 to 7 membered heterocyclyl” or “4 to 7 membered heterocycle” means aring with 4, 5, 6 or 7 ring atoms that may contain up to the maximumnumber of double bonds (aromatic or non-aromatic ring which is fully,partially or un-saturated) wherein at least one ring atom up to 4 ringatoms are replaced by a heteroatom selected from the group consisting ofsulfur (including —S(O)—, —S(O)₂—), oxygen and nitrogen (including═N(O)—) and wherein the ring is linked to the rest of the molecule via acarbon or nitrogen atom. Examples for a 4 to 7 membered heterocycles areazetidine, oxetane, thietane, furan, thiophene, pyrrole, pyrroline,imidazole, imidazoline, pyrazole, pyrazoline, oxazole, oxazoline,isoxazole, is oxazoline, thiazole, thiazoline, isothiazole,isothiazoline, thiadiazole, thiadiazoline, tetrahydrofuran,tetrahydrothiophene, pyrrolidine, imidazolidine, pyrazolidine,oxazolidine, is oxazolidine, thiazolidine, is othiazolidine,thiadiazolidine, sulfolane, pyran, dihydropyran, tetrahydropyran,imidazolidine, pyridine, pyridazine, pyrazine, pyrimidine, piperazine,piperidine, morpholine, tetrazole, triazole, triazolidine,tetrazolidine, diazepane, azepine or homopiperazine.

“9 to 11 membered heterobicyclyl” or “9 to 11 membered heterobicycle”means a heterocyclic system of two rings with 9 to 11 ring atoms, whereat least one ring atom is shared by both rings and that may contain upto the maximum number of double bonds (aromatic or non-aromatic ringwhich is fully, partially or un-saturated) wherein at least one ringatom up to 6 ring atoms are replaced by a heteroatom selected from thegroup consisting of sulfur (including —S(O)—, —S(O)₂—), oxygen andnitrogen (including ═N(O)—) and wherein the ring is linked to the restof the molecule via a carbon or nitrogen atom. Examples for a 9 to 11membered heterobicycle are indole, indoline, benzofuran, benzothiophene,benzoxazole, benzisoxazole, benzothiazole, benzisothiazole,benzimidazole, benzimidazoline, quinoline, quinazoline,dihydroquinazoline, quinoline, dihydroquinoline, tetrahydroquinoline,decahydroquinoline, isoquinoline, decahydroisoquinoline,tetrahydroisoquinoline, dihydroisoquinoline, benzazepine, purine orpteridine. The term 9 to 11 membered heterobicycle also includes spirostructures of two rings like 1,4-dioxa-8-azaspiro[4.5]decane or bridgedheterocycles like 8-aza-bicyclo[3.2.1]octane.

Preferably, L^(a)-S⁰ is selected from the group consisting of

-   -   wherein R is H; or C₁₋₄ alkyl; Y is NH; O; or S; and R¹, R^(1a),        R², R^(2a), R³, R^(3a), R⁴, X, X¹, X² have the meaning as        indicated above.

Even more preferred, L^(a)-S⁰ is selected from the group consisting of

wherein R has the meaning as indicated above.

At least one (up to four) hydrogen is replaced by a group L²-Z. In casemore than one group L²-Z is present each L² and each Z can be selectedindependently. Preferably, only one group L²-Z is present.

In general, S⁰ can be substituted with L²-Z at any position apart fromthe replacement of the hydrogen marked with an asterisk in the formulaeabove. Preferably, one to four of the hydrogen given by R, R¹ to R⁸directly or as hydrogen of the C₁₋₄ alkyl or further groups and ringsgiven by the definition of R and R¹ to R⁸ are replaced by L²-Z.

Furthermore, S⁰ may be optionally further substituted. In general, anysubstituent may be used as far as the cleavage principle is notaffected.

Preferably, one or more further optional substituents are independentlyselected from the group consisting of halogen; CN; COOR⁹; OR⁹; C(O)R⁹;C(O)N(R⁹R^(9a)); S(O)₂N(R⁹R^(9a)); S(O)N(R⁹R^(9a)); S(O)₂R⁹; S(O)R⁹;N(R⁹)S(O)₂N(R^(9a)R^(9b)); SR⁹; N(R⁹R^(9a)); NO₂; OC(O)R⁹;N(R⁹)C(O)R^(9a); N(R⁹)S(O)₂R^(9a); N(R⁹)S(O)R^(9a); N(R⁹)C(O)OR^(9a);N(R⁹)C(O)N(R^(9a)R^(9b)); OC(O)N(R⁹R^(9a)); T; C₁₋₅₀alkyl; C₂₋₅₀alkenyl; or C₂₋₅₀ alkynyl, wherein T; C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; andC₂₋₅₀ alkynyl are optionally substituted with one or more R¹⁰, which arethe same or different and wherein C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; and C₂₋₅₀alkynyl are optionally interrupted by one or more groups selected fromthe group consisting of T, —C(O)O—; —O—; —C(O)—; —C(O)N(R¹¹)—;—S(O)₂N(R¹¹)—; —S(O)N(R¹¹)—; —S(O)₂—; —S(O)—; —N(R¹¹)S(O)₂N(R^(11a))—;—S—; —N(R¹¹)—; —OC(O)R¹¹; —N(R¹¹)C(O)—; —N(R¹¹)S(O)₂—; —N(R¹¹)S(O)—;—N(R¹¹)C(O)O—; —N(R¹¹)C(O)N(R^(11a))—; and —OC(O)N(R¹¹R^(11a));

R⁹, R^(9a), R^(9b) are independently selected from the group consistingof H; T; and C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; or C₂₋₅₀ alkynyl, wherein T;C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; and C₂₋₅₀ alkynyl are optionally substitutedwith one or more R¹⁰, which are the same or different and wherein C₁₋₅₀alkyl; C₂₋₅₀ alkenyl; and C₂₋₅₀ alkynyl are optionally interrupted byone or more groups selected from the group consisting of T, —C(O)O—;—O—; —C(O)—; —C(O)N(R¹)—; —S(O)₂N(R)—; —S(O)N(R¹¹)—; —S(O)₂—; —S(O)—;—N(R¹¹)S(O)₂N(R^(11a))—; —S—; —N(R¹¹)—; —OC(O)R¹¹; —N(R¹¹)C(O)—;—N(R¹¹)S(O)₂—; —N(R¹¹)S(O)—; —N(R¹¹)C(O)O—; —N(R¹¹)C(O)N(R^(11a))—; and—OC(O)N(R¹¹R^(11a));

T is selected from the group consisting of phenyl; naphthyl; indenyl;indanyl; tetralinyl; C₃₋₁₀ cycloalkyl; 4 to 7 membered heterocyclyl; or9 to 11 membered heterobicyclyl, wherein T is optionally substitutedwith one or more R¹⁰, which are the same or different;

R¹⁰ is halogen; CN; oxo (═O); COOR¹²; OR¹²; C(O)R¹²; C(O)N(R¹²R^(12a));S(O)₂N(R¹²R^(12a)); S(O)N(R¹²R^(12a)); S(O)₂R¹²; S(O)R¹²;N(R¹²)S(O)₂N(R^(12a)R^(12b)); SR¹²; N(R¹²R^(12a)); NO₂; OC(O)R¹²;N(R¹²)C(O)R^(12a); N(R¹²)S(O)₂R^(12a); N(R¹²)S(O)R^(12a);N(R¹²)C(O)OR^(12a); N(R¹²)C(O)N(R^(12a)R^(12b)); OC(O)N(R¹²R^(12a)); orC₁₋₆ alkyl, wherein C₁₋₆ alkyl is optionally substituted with one ormore halogen, which are the same or different;

R¹¹, R^(11a), R¹², R^(12a), R^(12b) are independently selected from thegroup consisting of H; or C₁₋₆ alkyl, wherein C₁₋₆ alkyl is optionallysubstituted with one or more halogen, which are the same or different.

The term “interrupted” means that between two carbons a group isinserted or at the end of the carbon chain between the carbon andhydrogen.

L² is a single chemical bond or a spacer. In case L² is a spacer, it ispreferably defined as the one or more optional substituents definedabove, provided that L² is substituted with Z.

Accordingly, when L² is other than a single chemical bond, L²-Z isCOOR⁹; OR⁹; C(O)R⁹; C(O)N(R⁹R^(9a)); S(O)₂N(R⁹R^(9a)); S(O)N(R⁹R^(9a));S(O)₂R⁹; S(O)R⁹; N(R⁹)S(O)₂N(R^(9a)R^(9b)); SR⁹; N(R⁹R^(9a)); OC(O)R⁹;N(R)C(O)R^(9a); N(R)S(O)₂R^(9a); N(R⁹)S(O)R^(9a); N(R⁹)C(O)OR^(9a);N(R⁹)C(O)N(R^(9a)R^(9b)); OC(O)N(R⁹R^(9a)); T; C₁₋₅₀ alkyl; C₂₋₅₀alkenyl; or C₂₋₅₀ alkynyl, wherein T; C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; andC₂₋₅₀ alkynyl are optionally substituted with one or more R¹⁰, which arethe same or different and wherein C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; and C₂₋₅₀alkynyl are optionally interrupted by one or more groups selected fromthe group consisting of -T-, —C(O)O—; —O—; —C(O)—; —C(O)N(R¹¹)—;—S(O)₂N(R¹¹)—; —S(O)N(R¹¹)—; —S(O)₂—; —S(O)—; —N(R¹)S(O)₂N(R^(11a))—;—S—; —N(R¹¹)—; —OC(O)R¹¹; —N(R¹¹)C(O)—; —N(R¹¹)S(O)₂—; —N(R¹¹)S(O)—;—N(R¹¹)C(O)O—; —N(R¹¹)C(O)N(R^(11a))—; and —OC(O)N(R¹¹R^(11a));

R⁹, R^(9a), R^(9b) are independently selected from the group consistingof H; Z; T; and C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; or C₂₋₅₀ alkynyl, wherein T;C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; and C₂₋₅₀ alkynyl are optionally substitutedwith one or more R¹⁰, which are the same or different and wherein C₁₋₅₀alkyl; C₂₋₅₀ alkenyl; and C₂₋₅₀ alkynyl are optionally interrupted byone or more groups selected from the group consisting of T, —C(O)O—;—O—; —C(O)—; —C(O)N(R¹¹)—; —S(O)₂N(R¹¹)—; —S(O)N(R¹¹)—; —S(O)₂—; —S(O)—;—N(R¹¹)S(O)N(R^(11a))—; —S—; —N(R¹¹)—; —OC(O)R¹¹; —N(R¹¹)C(O)—;—N(R¹¹)S(O)₂—; —N(R¹¹)S(O)—; —N(R¹¹)C(O)O—; —N(R¹¹)C(O)N(R^(11a))—; and—OC(O)N(R¹¹R^(11a));

T is selected from the group consisting of phenyl; naphthyl; indenyl;indanyl; tetralinyl; C₃₋₁₀ cycloalkyl; 4 to 7 membered heterocyclyl; or9 to 11 membered heterobicyclyl, wherein t is optionally substitutedwith one or more R¹⁰, which are the same or different;

R¹⁰ is Z; halogen; CN; oxo (═O); COOR¹²; OR¹²; C(O)R¹²;C(O)N(R²R^(12a)); S(O) N(R¹²R^(12a)); S(O)N(R¹²R^(12a)); S(O)₂R¹²;S(O)R¹²; N(R¹²)S(O)₂N(R^(12a)R^(12b)); SR¹²; N(R¹²R^(12a)); NO₂;OC(O)R¹²; N(R¹²)C(O)R^(12a); N(R¹²)S(O)₂R^(12a); N(R¹²)S(O)R^(12a);N(R¹²)C(O)OR^(12a); N(R¹²)C(O)N(R^(12a)R^(12b)); OC(O)N(R¹²R^(12a)); orC₁₋₆ alkyl, wherein C₁₋₆ alkyl is optionally substituted with one ormore halogen, which are the same or different;

R¹¹, R^(11a), R¹², R^(12a), R^(12b) are independently selected from thegroup consisting of H; Z; or C₁₋₆ alkyl, wherein C₁₋₆ alkyl isoptionally substituted with one or more halogen, which are the same ordifferent;

provided that one of R⁹, R^(9a), R¹⁰, R¹¹, R^(11a), R¹², R^(12a),R^(12b) is Z.

Even more preferred general aromatic structures are listed below.

Preferably, the hGH polymer prodrug has the structure given in formula(A).

-   wherein in formula (A)-   NH-rhGH represents the rhGH residue attached to the transient linker    by forming a carbamate bond;-   R1, R2, R3, R4, and R5 are selected independently from hydrogen,    methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl,-   PEG represents a PEGylation residue attached to the transient    linker,-   and n=1 or 2, and-   X is selected from C1 to C8 alkyl or C1 to C12 heteroalkyl.

Preferably, the moiety

of formula (A) has the following structure

-   wherein each c is independently an integer from 250 to 750,    preferably an integer from 300 to 400 and more preferably 500.

The term “C1 to C12 heteroalkyl” means an alkyl chain having 1 to 12carbon atoms which are optionally interrupted by heteroatoms, functionalgroups, carbocycles or heterocycles as defined above. The term “C1 to C8alkyl” means C₁₋₈ alkyl as defined above.

In a preferred embodiment, in formula (A) L^(a) is represented by thecarbamate group attached to rhGH, G^(a) is represented by the aromaticoxygen group, the carbonyl attached to it, and the substituent attachedto the carbonyl as shown in formula I below.

More preferred structures are given by general formula I, which are partof the structure (A) within the general aromatic linker structure above:

and where preferred examples of formula I comprise:

More preferred aromatic structures of formula II, which are part of thestructure (A) within the general aromatic linker structure above:

and where preferred examples of formula II comprise:

More preferred structures of formula III, which are part of thestructure (A) within the general aromatic linker structure above,wherein PEG-X is

and PEG-W includes the following substituent groups:

Further examples of preferred hGH polymer prodrug conjugates are shownbelow:

R is selected from hydrogen, methyl, ethyl, propyl and butyl,X is selected from C1 to C8 alkyl or C1 to C12 heteroalkyl.

Also in the preferred and more preferred embodiments PEG meanspreferably the rest of S⁰, comprising at least S¹, S², BS¹ andoptionally BS².

In a preferred embodiment prodrugs comprised in the compositions of thepresent invention are selected from the group consisting of

wherein m is an integer from 200 to 250 and n is an integer from 100 to125;

wherein n is an integer from 400 to 500;

wherein n is an integer from 400 to 500; and

wherein n is an integer from 400 to 500.

Preferably, PEGylation of the hGH moiety occurs at one or more of thelysines selected from the group consisting of Lys158, Lys145, Lys38,Lys140 and Lys70. More preferably, PEGylation of the hGH moiety occursmainly at positions Lys158, Lys145, Lys38 and Lys140, even morepreferably mainly at positions Lys158, Lys145 and Lys38.

Preferably, at least 30% of all growth hormone moieties of thecomposition of the present invention are PEGylated at position Lys158.

hGH polymer prodrugs comprised in the dry compositions of the presentinvention can be prepared by methods known in the art. However,especially for compounds of formula (AA1) it is preferred to build upthe prodrug molecule in a convergent synthesis by providing a firstprecursor molecule comprising one or more thiol groups and an activatedcarbonate group and a second precursor molecule comprising a maleimidegroup to react in an addition reaction resulting in the formation of athio succinimide group and to react that combined precursor moleculewith hGH to yield a compound of formula (AA1).

Accordingly, a method for the preparation of a compound of formulahGH-NH—C(O)O—S⁰ (AA1), wherein S⁰ has the meaning as indicated above andcomprises at least one group

comprises the following steps:

-   -   (a) reacting a compound of formula ROC(O)O—S^(0′)—SH (AA1′) with        a compound of formula

-   -   -   wherein R is a suitable rest for an activated carbonate            group and wherein S^(0′) and S^(0″″) are selected to yield            S⁰ comprising the at least one group

resulting in a compound of formula ROC(O)O—S⁰, and

-   -   (b) reacting the compound of formula ROC(O)O—S⁰ with hGH-NH₂,        wherein hGH-NH₂ represents hGH with one of its primary amino        groups to yield a compound of formula (AA1).

Suitable R groups for the carbonate functional groups includesubstituted alkyl or carbocyclic or heterocyclic, like aryl orcycloalkyl, groups like the pentafluorophenyl or NHS group.

The stable composition comprised of rhGH polymer prodrug as describedabove is a dry composition with a shelf-life of preferably at least 6months, more preferably at least 1 year, more preferably at least 2years, when stored at temperatures ranging from −80° C. up to 25° C.,preferably ranging from 2 to 25° C. The preferred method of drying therhGH polymer prodrug composition is lyophilization.

Thus a further aspect of the present invention is a composition which isdried by lyophilisation. Another aspect is a composition of the presentinvention which is stable for at least 1 year when stored attemperatures ranging from −80° C. up to 25° C., preferably ranging from2-25° C. More preferably the temperature is from 2° C. to 8° C. Anotherpreferred temperature range is from 15° C. to 25° C.

The dry composition of rhGH polymer prodrug according to the presentinvention comprises one or more lyoprotectants. Exemplary lyoprotectantsinclude sugars, such as sucrose or trehalose; amino acids such asmonosodium glutamate or histidine or arginine; methylamines such asbetaine; lyotropic salts such as magnesium sulfate; polyols such astrihydric or higher sugar alcohols, e.g. glycerin, erythritol, glycerol,arabitol, xylitol, sorbitol, and mannitol; ehthylene glycol; propyleneglycol; polyethylene glycol; pluronics; hydroxyalkyl starches, e.g.hydroxyethyl starch (HES), and combinations thereof. Preferably, thelyoprotectants include sugars, such as sucrose or trehalose; amino acidssuch as monosodium glutamate or histidine; methylamines such as betaine;lyotropic salts such as magnesium sulfate; polyols such as trihydric orhigher sugar alcohols, e.g. glycerin, erythritol, glycerol, arabitol,xylitol, sorbitol, and mannitol; ehthylene glycol; propylene glycol;polyethylene glycol; pluronics; hydroxyalkyl starches, e.g. hydroxyethylstarch (HES), and combinations thereof.

The preferred lyoprotectant is a non-reducing sugar, such as trehaloseor sucrose, especially trehalose.

The lyoprotectant is preferably added to the composition before thedrying step in a “lyoprotecting amount” which means that, followingdrying of the protein prodrug in the presence of the lyoprotectingamount of the lyoprotectant, the protein prodrug essentially retains itsphysical and chemical stability and integrity upon drying and storage.

The dry compositions of rhGH polymer prodrug according to the presentinvention may contain one or more excipients. Excipients used inparenteral compositions may be categorized as, for example, bufferingagents, isotonicity modifiers, preservatives, stabilizers,anti-adsorption agents, oxidation protection agents, or other auxiliaryagents. However, in some cases, one excipient may have dual or triplefunctions. The dry composition may contain one or more than one of thefollowing excipients:

-   -   (i) Buffering agents: physiologically tolerated buffers to        maintain pH in a desired range, such as sodium phosphate,        bicarbonate, succinate, histidine, citrate and acetate,        sulphate, nitrate, chloride, pyruvate. Antacids such as Mg(OH)₂        or ZnCO₃ may be also used. Buffering capacity may be adjusted to        match the conditions most sensitive to pH stability    -   (ii) Isotonicity modifiers: to minimize pain that can result        from cell damage due to osmotic pressure differences at the        injection depot. Glycerin and sodium chloride are examples.        Effective concentrations can be determined by osmometry using an        assumed osmolality of 285-315 mOsmol/kg for serum    -   (iii) Preservatives: multidose parenteral preparations require        the addition of preservatives at sufficient concentration to        minimize risk of patients becoming infected upon injection and        corresponding regulatory requirements have been established.        Typical preservatives include m-cresol, phenol, methylparaben,        ethylparaben, propylparaben, butylparaben, chlorobutanol, benzyl        alcohol, phenylmercuric nitrate, thimerosol, sorbic acid,        potassium sorbate, benzoic acid, chlorocresol, and benzalkonium        chloride    -   (iv) Stabilizers: Stabilisation is achieved by strengthening of        the protein-stabilising forces, by destabilisation of the        denatured state, or by direct binding of excipients to the        protein. Stabilizers may be amino acids such as alanine,        arginine, aspartic acid, glycine, histidine, lysine, proline,        sugars such as glucose, sucrose, trehalose, polyols such as        glycerol, mannitol, sorbitol, salts such as potassium phosphate,        sodium sulphate, chelating agents such as EDTA, hexaphosphate,        ligands such as divalent metal ions (zinc, calcium, etc.), other        salts or organic molecules such as phenolic derivatives. In        addition, oligomers or polymers such as cyclodextrins, dextran,        dendrimers, PEG or PVP or protamine or HSA may be used    -   (v) Anti-adsorption agents: Mainly ionic or inon-ionic        surfactants or other proteins or soluble polymers are used to        coat or adsorb compentitively to the inner surface of the        composition's or composition's container. Eg, poloxamer        (Pluronic F-68), PEG dodecyl ether (Brij 35), polysorbate 20 and        80, dextran, polyethylene glycol, PEG-polyhistidine, BSA and HSA        and gelatines. Chosen concentration and type of excipient        depends on the effect to be avoided but typically a monolayer of        surfactant is formed at the interface just above the CMC value    -   (vi) Cryoprotectants: During freeze- or spray drying, excipients        may counteract the destabilising effects caused by hydrogen bond        breaking and water removal. For this purpose sugars and polyols        may be used but corresponding positive effects have also been        observed for surfactants, amino acids, non-aqueous solvents, and        other peptides. Trehalose is particularly efficient at reducing        moisture-induced aggregation and also improves thermal stability        potentially caused by exposure of protein hydrophobic groups to        water. Mannitol and sucrose may also be used, either as sole        lyo/cryoprotectant or in combination with each other where        higher ratios of mannitol:sucrose are known to enhance physical        stability of a protein in a dry composition. Mannitol may also        be combined with trehalose. Trehalose may also be combined with        sorbitol or sorbitol used as the sole protectant. Starch or        starch derivatives may also be used    -   (vii) Oxidation protection agents: antioxidants such as ascorbic        acid, ectoine, methionine, glutathione, monothioglycerol, morin,        polyethylenimine (PEI), propyl gallate, vitamin E, chelating        agents such as citric acid, EDTA, hexaphosphate, thioglycolic        acid    -   (viii) Other auxiliary agents: such as wetting agents, viscosity        modifiers, antibiotics. Acids and bases such as hydrochloric        acid and sodium hydroxide are auxiliary agents necessary for pH        adjustment during manufacture.

In one embodiment of the present invention, the dry composition of rhGHpolymer prodrug is provided as a single dose, meaning that the containercomprising the dry composition of rhGH polymer prodrug comprises onetherapeutic dose.

Thus, in another aspect of the present invention the composition isprovided as a single dose composition.

In another embodiment, the dry composition of rhGH polymer prodrugcontains multiple doses, meaning that the container comprising the drycomposition of rhGH polymer prodrug contains more than one therapeuticdose. Preferably, a multiple dose composition contains at least 2 doses,such as from 2 to 12 doses of hGH polymer prodrug and preferably atleast 4 doses.

Thus, in another aspect of the present invention the composition isprovided as a multiple dose composition.

In a further aspect, the composition according to the present inventionis characterized in that it contains one or more additional biologicallyactive agents, either in its free form or as a prodrug, and wherein theone or more additional biologically active agents are selected from thegroup consisting of IGF-1, ghrelin or ghrelin-like compounds,gonadotropin releasing hormone agonists and/or analogs, growth hormonereleasing factor and analogs, gonadal steroids, antiandrogens,non-steroidal aromatase inhibitors, HIV combination therapy, free fattyacid regulators, anabolic steroids, estrogen agonists/antagonists,propranolol, appetite suppressants osteroporosis drugs includingbisphosphonates, bone formation agents, estrogens, parathyroid hormones,and selective receptor modulators, and/or anti-diabetic drugs such asinsulin, thiazolidinediones, sulfonyl ureas, incretin memetics,meglitinides, biguanides, alpha-glucosidase inhibitors and amylinanalogues. Prodrugs used for additional biologically active agents arepreferably also transient polymer prodrugs and especially prodrugs asdescribed herein for rhGH. Preferably, the additional biologicallyactive agents are contained in there free from.

Prior to applying the dry composition of the rhGH polymer prodrugcomposition to a patient in need thereof, the dry composition isreconstituted. Reconstitution can take place in the container in whichthe dry composition of rhGH polymer prodrug is provided, such as in avial, syringe, dual-chamber syringe, ampoule, and cartridge, or the drycomposition of rhGH polymer prodrug may be transferred to a differentcontainer and is then reconstituted. Reconstitution is done by adding apredefined amount of reconstitution solution to the lyophilisate. Thereconstitution solutions are sterile liquids, such as water or buffer,which may contain further additives, such as preservatives and/orantimicrobials. If the rhGH polymer prodrug composition is provided assingle dose, the reconstitution solution may contain one or more thanone preservative and/or antimicrobial. Preferably, it is sterile water.If the rhGH polymer prodrug composition is a multiple dose composition,it is preferred that the reconstitution solution contains one or morepreservative and/or antimicrobial, such as, for example, benzylalcoholand cresol.

The reconstituted composition preferably comprises

rhGH polymer prodrug 10-300 mg/ml succinic acid 5-50 mM trehalosedihydrate 25-850 mg/ml,and has a pH ranging from pH 4.5 to pH 6.

More preferably, the reconstituted composition comprises

rhGH polymer prodrug 10-300 mg/ml succinic acid 5-50 mM trehalosedihydrate 30-150 mg/ml,and has a pH ranging from pH 4.5 to pH 6.

Even more preferably, the reconstituted composition comprises

rhGH polymer prodrug 10-300 mg/ml succinic acid 5-50 mM trehalosedihydrate 50-100 mg/ml,and has a pH ranging from pH 4.5 to pH 6.

Most preferably, the reconstituted composition comprises

rhGH polymer prodrug 30-60 mg/ml succinic acid 10 mM trehalose dihydrate70-85 mg/ml,and has a pH ranging from pH 4.5 to pH 5.5.

Optionally, the reconstituted composition comprises one or morepreservative and/or antimicrobial.

Optionally, the reconstituted composition comprises one or moreexcipient.

After reconstitution, a single dose composition of rhGH polymer prodrughas a volume of not more than 4 ml, such as from about 0.5 to about 3.5ml.

In a reconstituted, multi-dose composition of rhGH polymer prodrug, eachdose has a volume of not more than 4 ml, such as from about 0.5 to about3.5 ml. The individual therapeutic doses of such multiple dosecomposition of rhGH polymer prodrug can either be used for differentpatients in need thereof or can be used for one patient, whereas afterthe application of the first dose the remaining doses are stored untilneeded. In the latter case, the reconstituted rhGH polymer prodrug isstable for at least 2 weeks, preferably for at least 4 weeks, and morepreferably for at least 6 weeks, when stored at 2-8° C.

Preferably, the hGH polymer prodrug is sufficiently dosed in thecomposition to provide therapeutically effective amount of hGH for atleast three days in one application. More preferably, the oneapplication of the hGH polymer prodrug provides therapeuticallyeffective amount of hGH for four days, even more preferably for fivedays and most preferably for one week.

In another aspect of the present invention the composition is comprisedin a container, such as a vial, syringe, dual-chamber syringe, ampoule,and cartridge. Preferably the container is a dual-chamber syringe.Especially the dry composition according to the present invention isprovided in a first chamber of the dual-chamber syringe andreconstitution solution is provided in a second chamber of thedual-chamber syringe.

Another aspect of the present invention is a method of manufacturing acomposition according to the present invention, comprising the steps of

-   (i) admixing the rhGH polymer prodrug with one or more    lyoprotectants and optionally one or more excipients,-   (ii) transferring amounts of the mixture from step (i) equivalent to    the desired number of dosages into suitable chambers of a container,-   (iii) drying the mixture, and-   (iv) sealing the container.

Preferably, the method of manufacturing a composition of the presentinvention comprises the steps of

-   (i) admixing the rhGH polymer prodrug with trehalose dihydrate and    succinic acid to yield a composition comprising

rhGH polymer prodrug 10-300 mg/ml succinic acid 5-50 mM trehalosedihydrate 25-850 mg/ml,

-   (ii) adjusting the pH of the composition of step (i) to a pH ranging    from pH 4.0 to pH 6.5 with tris,-   (iii) transferring amounts of the mixture from step (i) equivalent    to the desired number of dosages into suitable chambers of a    container,-   (iv) drying the mixture, and-   (v) sealing the container;    whereas the order of steps (ii) and (iii) may be changed.

Preferably, the composition in step (i) comprises

rhGH polymer prodrug 10-300 mg/ml succinic acid 5-50 mM trehalosedihydrate 50-100 mg/ml.

More preferably, the composition in step (i) comprises

rhGH polymer prodrug 30-60 mg/ml succinic acid 10 mM trehalose dihydrate70-85 mg/ml.

The number of chambers may depend on the number of dosages. If only onedosage is intended the container may comprise one, two or more chambers.

Preferably, step (iii) is done by lyophilization.

Preferably, in the method described above the container is adual-chamber syringe having a first chamber with the mixture comprisingthe hGH polymer prodrug composition, the method further comprising thestep of

-   -   filling a second chamber with a reconstitution solution before        sealing the container.

Another aspect is a kit of parts. In case the administration device issimply a hypodermic syringe then the kit may comprise the syringe, aneedle, a vial containing the reconstitution solution, and a vial orampoule containing the dry hGH polymer prodrug composition for use withthe syringe. Optionally, the kit of parts comprises a safety device forthe needle which can be used to cap or cover the needle after use toprevent injury.

In more preferred embodiments, the injection device is other than asimple hypodermic syringe and so the separate container is adapted toengage with the injection device such that in use the liquid compositionin the container is in fluid connection with the outlet of the injectiondevice. Most preferably, the separate container is a dual-chambersyringe. Examples of administration devices include but are not limitedto hypodermic syringes and pen injector devices. Particularly preferredinjection devices are the pen injectors in which case the container is acartridge, preferably a disposable cartridge.

A preferred kit of parts comprises a needle and a container containingthe composition according to the present invention and optionallyfurther containing a reconstitution solution, the container beingadapted for use with the needle. Preferably, the container is adual-chamber syringe.

An additional aspect of the present invention relates to the method ofadministration of a reconstituted rhGH polymer prodrug composition. TherhGH polymer prodrug composition can be administered by methods ofinjection or infusion, including intradermal, subcutaneous,intramuscular, intravenous, intraosseous, and intraperitoneal.

A further aspect is a method of preparing a reconstituted compositioncomprising a therapeutically effective amount of a rhGH polymer prodrug,one or more lyoprotectants and optionally one or more pharmaceuticallyacceptable excipients, wherein the growth hormone is transiently linkedto a polymer carrier, the method comprising the step of

-   -   Contacting the composition of the present invention with a        reconstitution solution.

Another aspect is a reconstituted composition comprising atherapeutically effective amount of a rhGH polymer prodrug, one or morelyoprotectants and optionally one or more pharmaceutically acceptableexcipients, wherein the growth hormone is transiently linked to apolymer carrier obtainable by the method above.

In one embodiment of the present invention the dry composition ofpolymer rhGH prodrug does not only comprise a rhGH polymer prodrug, alyoprotectant and, optionally, one or more excipients, but also otherbiologically active moieties, either in their free form or as prodrugs.Such biologically active moieties are, for example:

-   -   IGF-1    -   ghrelin or ghrelin-like compounds,    -   gonadotropin releasing hormone analog such as, for example,        triptorelin or antiandrogen, such as, for example, cyproterone        acetate or non-steroidal aromatase inhibitors, such as, for        example, letrozole    -   HIV combination therapy    -   free fatty acid regulator, e.g. acipimox    -   anabolic steroid, eg oxandrolone    -   estrogen agonist/antagonist    -   propranolol    -   appetite suppressant, e.g. sibutramine

If the dry composition of hGH polymer prodrug of the present inventioncomprises other biologically active moieties in the form of a prodrug,then it is preferred that said other biologically active moieties aretransiently linked to a polymer. It is also preferred that the transientlinkage comprises any of the before described linkers.

In an alternative embodiment, the rhGH polymer prodrug compositionaccording to the present invention is combined with a secondbiologically active compound in such way that the rhGH polymer prodrugis administered to a patient in need thereof first, followed by theadministration of the second compound. Alternatively, the rhGH polymerprodrug composition is administered to a patient in need thereof afteranother compound has been administered to the same patient.

In a preferred embodiment, the composition of dry rhGH polymer prodrughas the following composition (based on the total weight of thecomposition):

rhGH polymer prodrug 1.1-92.1% (w/w) succinic acid 0.1-14.4% (w/w)trehalose, optionally as dihydrate 7.3-98.7% (w/w) tris 0.01-25.4% (w/w)

More preferably, the composition of dry rhGH polymer prodrug has thefollowing composition (based on the total weight of the composition):

rhGH polymer prodrug 7.8-85.5% (w/w) succinic acid 0.1-8.9% (w/w)trehalose, optionally as dihydrate 13.6-90.3% (w/w) tris 0.03-15.7%(w/w)

Most preferably, the composition of dry rhGH polymer prodrug has thefollowing composition (based on the total weight of the composition):

rhGH polymer prodrug 25.3-46.5% (w/w) succinic acid 0.8-1.2% (w/w)trehalose, optionally as dihydrate 52.4-72.8% (w/w) tris 0.4-2.3% (w/w)

Preferably, the composition of rhGH polymer prodrug as described aboveis lyophilized. Preferably, it is lyophilized in a first chamber of adual-chamber syringe, of which second chamber is filled withreconstitution solution. In one embodiment, the reconstitution solutionis sterile water containing 0.7-1.1% benzylalcohol, more preferably 0.9%bezylalcohol. In another embodiment, the reconstitution solutioncontains 0.2-0.4% cresol, more preferably 0.3% cresol. Preferably, thereconstitution solution is sterile water.

Any of the above described compositions of rhGH polymer prodrugs areused for treating or preventing diseases or disorders, which can betreated by rhGH, such as growth hormone deficiency (GHD), adult onsetgrowth hormone deficiency, Turner syndrome, Prader-Willi syndrome, shortbowel syndrome, chronic renal insufficiency, small for gestational age(SGA), AIDS wasting, anti-ageing, rheumatoid arthritis, idiopathic smallstature, short stature homeobox gene and somatopause. Included is alsoshort stature associated with prolonged steroid use, Aarskog's syndrome,among others.

Also included are chronic renal disease, juvenile rheumatoid arthritis;cystic fibrosis, HIV-infection in children receiving HAART treatment(HIV/HALS children); short stature in children born with very low birthweigth (VLBW) but SGA; skeletal dysplasia; hypochondroplasia;achondroplasia; idiopathic short stature (ISS); GHD in adults; fracturesin or of long bones, such as tibia, fibula, femur, humerus, radius,ulna, clavicula, matacarpea, matatarsea, and digit; fractures in or ofspongious bones, such as the scull, base of hand, and base of foot;patients after tendon or ligament surgery in e.g. hand, knee, orshoulder; distraction oteogenesis; disorders resulting from hip ordiscus replacement, meniscus repair, spinal fusions or prothesisfixation, such as in the knee, hip, shoulder, elbow, wrist or jaw;disorders resulting from fixing of osteosynthesis material, such asnails, screws and plates; non-union or mal-union of fractures; disordersresulting from osteatomia, e.g. from tibia or 1^(st) toe; disordersresulting from graft implantation; articular cartilage degeneration inknee caused by trauma or arthritis; osteoporosis in patients with Turnersyndrome; osteoporosis in men; adult patients in chronic dialysis(APCD); malnutritional associated cardiovascular disease in APCD;reversal of cachexia in APCD; cancer in APCD; chronic abstractivepulmonal disease in APCD; HIV in APCD; elderly with APCD; chronic liverdisease in APCD, fatigue syndrome in APCD; Crohn's disease; impairedliver function; males with HIV infections; short bowel syndrome; centralobesity; HIV-associated lipodystrophy syndrome (HALS); male infertility;patients after major elective surgery, alcohol/drug detoxification orneurological trauma; aging; frail elderly; osteo-arthritis;traumatically damaged cartilage; erectile dysfunction; fibromyalgia;memory disorders; depression; traumatic brain injury; subarachnoidhaemorrhage; very low birth weigth; metabolic syndrome; glucocorticoidmyopathy; and short stature due to glucucorticoid treatment in children.

EXAMPLES Example 1: Synthesis of hGH Polymer Prodrug

hGH polymer prodrug (1) with c˜500 was synthesized as described in WO-A2009/133137.

Example 2: Stability Testing of Compositions Containing hGH PolymerProdrug

Five different lyophilized compositions (C1, C2, C3, C4, and C5) of hGHpolymer prodrug were prepared. Each composition contained amounts of hGHpolymer prodrug 1 to yield a concentration of 5 mg/ml afterreconstitution. Formulations were placed upright in an incubator set to40° C./75% RH. After 17 days, one vial per formulation was removed fromthe respective incubator, reconstituted with sterile water for injectionand subjected to analysis.

hGH polymer prodrug Trehalose Mannitol Glycine (mg/mL) Buffer Base pH(mg/mL) (mg/mL) (mg/mL) C1 5 Succinic acid Tris 4.0 92 — — (10 mM) C2 5Succinic acid Tris 4.0 10 40 — (10 mM) C3 5 Succinic acid Tris 4.0 — 4620 (10 mM) C4 5 Succinic acid Tris 6.0 10 40 — (10 mM) C5 5 Phosphate (5mM) Sodium 6.5 10 40 — hydroxide Free hGH, Visual % of total PeptidePeptide Peptide Peptide inspection integrated area mapping (% mapping (%mapping (% mapping (% before/after for free hGH impurity) impurity)impurity) impurity) reconstitution peak isoaspartate succinimidedeamidation oxidation C1 Intact white 0.15 (t = 0); 1.89 (t = 0); 1.75(t = 0); 2.0 (t = 0); 1.25 (t = 0); cake/clear 0.35 (t = 17 d) 3.22 (t =17 d) 2.79 (t = 17 d) 1.9 (t = 17 d) 1.64 (t = 17 d) colorless C2 Intactwhite 0.14 (t = 0); 1.82 (t = 0); 2.24 (t = 0); 1.8 (t = 0); 1.39 (t =0); cake/clear 0.51 (t = 17 d) 5.68 (t = 17 d) 5.08 (t = 17 d) 1.8 (t =17 d) 1.65 (t = 17 d) colorless C3 Intact white 0.11 (t = 0); 1.79 (t =0); 2.23 (t = 0); 2.0 (t = 0); 1.25 (t = 0); cake/clear 0.65 (t = 17 d)8.69 (t = 17 d) 8.09 (t = 17 d) 2.3 (t = 17 d) 1.22 (t = 17 d) colorlessC4 Intact white 0.39 (t = 0); 1.59 (t = 0); 1.90 (t = 0); 2.6 (t = 0);1.16 (t = 0); cake/clear 1.46 (t = 17 d) 2.16 (t = 17 d) 2.19 (t = 17 d)2.8 (t = 17 d) 1.23 (t = 17 d) colorless C5 Intact white 0.47 (t = 0);1.42 (t = 0); 1.69 (t = 0); 3.1 (t = 0); 1.94 (t = 0); cake/clear 1.04(t = 17 d) 1.94 (t = 17 d) 1.71 (t = 17 d) 3.2 (t = 17 d) 1.90 (t = 17d) colorless

Example 3: Stability Testing of Compositions Containing hGH PolymerProdrug

Three different lyophilized compositions (C6, C7, and C8) of hGH polymerprodrug were prepared. Each composition contained amounts of hGH polymerprodrug 1 to yield a concentration of 30 mg/ml after reconstitution.Formulations were placed upright in an incubator set to 40° C./75% RHand an incubator set to 2-8° C., respectively. At each time point, onevial per formulation was removed from the respective incubator,reconstituted with sterile water for injection and subjected toanalysis.

hGH polymer prodrug Trehalose (mg/mL) Buffer Base pH (mg/mL) C6 30Succinic acid (10 mM) Tris 4.0 92 C7 30 Succinic acid (10 mM) Tris 5.092 C8 30 Phosphate (10 mM) Sodium 6.0 92 hydroxide Free hGH after nweeks at 40° C./ Free hGH after n Visual 75% RH, weeks at 2-8° C.,inspection % of total % of total before/after integrated area integratedarea reconstitution for free hGH peak for free hGH peak C6 Intact white0.4 (n = 0); 0.4 (n = 0); cake/clear 0.6 (n = 2); 0.4 (n = 4); colorless0.7 (n = 4); 0.4 (n = 9) 0.9 (n = 9) C7 Intact white 0.6 (n = 0); 0.6 (n= 0); cake/clear 0.9 (n = 2); 0.6 (n = 4); colorless 1.0 (n = 4); 0.6 (n= 9) 1.4 (n = 9) C8 Intact white 0.7 (n = 0); 0.7 (n = 0); cake/clear1.1 (n = 2); 0.7 (n = 4); colorless 1.2 (n = 4); 0.7 (n = 9) 1.6 (n = 9)

Example 4: Stability Testing of Compositions Containing hGH PolymerProdrug

A lyophilized composition (C9) of hGH polymer prodrug was prepared. Thecomposition contained amounts of hGH polymer prodrug 1 to yield aconcentration of 30 mg/ml after reconstitution. Formulation was placedupright in an incubator set to 40° C./75% RH. After each time point, onevial was removed from the respective incubator, reconstituted withsterile water for injection and subjected to analysis.

hGH polymer Trehalose prodrug dihydrate (mg/mL) Buffer Base pH (mg/mL))C9 at 40° C. 30 Succinic Tris 5.0 85 acid (10 mM) Free hGH, % of totalPeptide Peptide Peptide Peptide integrated mapping (% mapping (% mapping(% mapping (% area for impurity) impurity) impurity) impurity) Visualinspection free hGH, Isoaspartate Succinimide Deamidation Oxidationbefore/after peak after n (after n (after n (after n (after nreconstitution weeks weeks) weeks) weeks) weeks) C9 at Intact white 0.6(n = 0); 1.8 (n = 2); 0.8 (n = 2); 4.4 (n = 2); 2.1 (n = 2); 40° C.cake, no cracking 0.9 (n = 2); 2.0 (n = 4); 0.9 (n = 4); 4.2 (n = 4);3.1 (n = 4); around 1.0 (n = 4); 2.1 (n = 9) 1.2 (n = 9) 5.3 (n = 9) 3.2(n = 9) meniscus, no 1.4 (n = 9) loose pieces, no pull back from vial

Example 5: Stability Testing of Compositions Containing hGH PolymerProdrug

A lyophilized composition (C20) of hGH polymer prodrug was prepared. Thecomposition contained amounts of hGH polymer prodrug 1 to yield aconcentration of 30 mg/ml after reconstitution. The formulations wereplaced upright in an incubator set to 5±3° C. and an incubator set to25±2° C., respectively. After each time point, one vial was removed fromthe respective incubator, reconstituted with sterile water for injectionand subjected to analysis.

hGH polymer prodrug Trehalose dihydrate (mg/mL) Buffer Base (mg/mL) pHafter n months C20 at 30 Succinic acid (10 mM) Tris 85 5.1 (n = 0); 5°C. 5.1 (n = 1); 5.0 (n = 3); 5.0 (n = 4); 5.1 (n = 6); 5.1 (n = 9); 4.9(n = 12); 5.1 (n = 15); 5.1 (n = 18) C20 at 30 Succinic acid (10 mM)Tris 85 5.1 (n = 0); 25° C. 5.1 (n = 1); 5.1 (n = 3); 5.0 (n = 4); 5.1(n = 6); 5.0 (n = 9); 5.0 (n = 12); 5.0 (n = 15); 5.0 (n = 18) Free hGH,% of total Peptide Peptide Peptide Peptide integrated mapping (% mapping(% mapping (% mapping (% Visual area for free impurity) impurity)impurity) impurity) inspection hGH peak isoaspartate succinimidedeamidation oxidation before/after after n after n after n after n aftern reconstitution months months months months months C20 Intact white 1.4(n = 0); 1.9 (n = 0); 0.8 (n = 0); 4.0 (n = 0); 2.9 (n = 0); atcake/clear 1.4 (n = 1); 1.9 (n = 1); 0.7 (n = 1); 4.0 (n = 1); 3.4 (n =1); 5° C. colorless 1.4 (n = 3); 1.8 (n = 3); 0.8 (n = 3); 3.9 (n = 3);2.7 (n = 3); 1.4 (n = 4); 1.8 (n = 4); 0.8 (n = 4); 4.0 (n = 4); 2.5 (n= 4); 1.4 (n = 6); 1.9 (n = 6); 0.7 (n = 6); 4.0 (n = 6); 2.5 (n = 6);1.4 (n = 9); 1.6 (n = 9); 1.9 (n = 9); 3.9 (n = 9); 2.5 (n = 9); 1.5 (n= 12); 1.9 (n = 12); 1.2 (n = 12); 5.1 (n = 12); 2.7 (n = 12); 1.4 (n =15); 1.8 (n = 15); 1.3 (n = 15); 3.9 (n = 15); 2.7 (n = 15); 1.4 (n =18) 1.8 (n = 18) 1.4 (n = 18) 3.9 (n = 18) 2.9 (n = 18) C20 Intact white1.4 (n = 0); 1.9 (n = 0); 0.8 (n = 0); 4.0 (n = 0); 2.9 (n = 0); atcake/clear 1.4 (n = 1); 1.9 (n = 1); 0.7 (n = 1); 4.3 (n = 1); 3.3 (n =1); 25° C. colorless 1.5 (n = 3); 1.9 (n = 3); 0.8 (n = 3); 4.0 (n = 3);3.1 (n = 3); 1.5 (n = 4); 1.9 (n = 4); 0.9 (n = 4); 4.0 (n = 4); 2.7 (n= 4); 1.5 (n = 6); 2.0 (n = 6); 0.8 (n = 6); 3.9 (n = 6); 2.7 (n = 6);1.7 (n = 9); 1.8 (n = 9); 1.0 (n = 9); 3.8 (n = 9); 2.7 (n = 9); 1.7 (n= 12); 2.1 (n = 12); 1.5 (n = 12); 4.8 (n = 12); 2.9 (n = 12); 1.7 (n =15); 2.1 (n = 15); 1.7 (n = 15); 3.8 (n = 15); 3.1 (n = 15); 1.7 (n =18) 2.3 (n = 18) 1.6 (n = 18) 4.4 (n = 18) 3.3 (n = 18)

Example 6: Stability Testing of Compositions Containing hGH PolymerProdrug

A lyophilized composition (C10) of hGH polymer prodrug was prepared. Thecomposition contained amounts of hGH polymer prodrug 1 to yield aconcentration of 44 mg/ml after reconstitution. The formulations wereplaced upright in an incubator set to 40±2° C. at a relative humidity of75±5%. After each time point, one vial was removed from the respectiveincubator, reconstituted with sterile water for injection and subjectedto analysis.

hGH polymer prodrug (mg/mL) Buffer Base pH Trehalose (mg/mL) C10 44Succinic acid Tris 5.0 75 (10 mM) Free hGH, Peptide % of total mappingintegrated Peptide Peptide Peptide (% area for mapping (% mapping (%mapping (% impurity) Visual inspection free hGH impurity) impurity)impurity) oxidation before/after peak after n isoaspartate succinimidedeamidation after n reconstitution weeks after n weeks after n weeksafter n weeks weeks C10 Intact white 0.3 (n = 0); 1.7 (n = 0); 1.1 (n =0); 4.8 (n = 0); 1.9 (n = 0); at cake, no cracking 0.7 (n = 4); 2.0 (n =4); 1.2 (n = 4); 4.8 (n = 4); 2.0 (n = 4); 40° C. around 1.1 (n = 8);2.2 (n = 8) 1.3 (n = 8) 4.8 (n = 8) 2.1 (n = 8) meniscus, no loosepieces, no pull back from vial

Example 7: Stability Testing of Compositions Containing hGH PolymerProdrug

A lyophilized composition (C11) of hGH polymer prodrug was prepared. Thecomposition contained amounts of hGH polymer prodrug 1 to yield aconcentration of 52 mg/ml after reconstitution. The formulations wereplaced upright in an incubator set to 40±2° C. at a relative humidity of75±5%. After each time point, one vial was removed from the respectiveincubator, reconstituted with sterile water for injection and subjectedto analysis.

hGH polymer prodrug (mg/mL) Buffer Base pH Trehalose (mg/mL) C11 52Succinic acid Tris 5.0 74 (10 mM) Free hGH, Peptide Peptide % of totalPeptide mapping mapping integrated mapping (% Peptide (% (% Visual areafor impurity) mapping (% impurity) impurity) inspection free hGHisoaspartate impurity) deamidation oxidation before/after peak afterafter n succinimide after after n reconstitution n weeks weeks after nweeks n weeks weeks C11 Intact white 2.0 (n = 0); 1.3 (n = 0); 0.8 (n =0); 4.2 (n = 0); 1.3 (n = 0); at cake, no 2.6 (n = 4); 1.6 (n = 4); 1.0(n = 4); 4.2 (n = 4); 1.5 (n = 4); 40° C. cracking 2.9 (n = 8); 1.7 (n =8) 1.1 (n = 8) 3.9 (n = 8) 1.8 (n = 8) around meniscus, no loose pieces,no pull back from vial

Methods:

Lyophilized compositions: Ultra-/diafiltration or dialysis was employedto obtain a concentrated aqueous solution containing hGH polymer prodrugfrom column eluate. Buffer exchange against buffer containing succinateor phosphate pH-adjusted by Tris or sodium hydroxide, respectively, andtrehalose dihydrate and/or mannitol and/or glycine, was performed toobtain an aqueous buffered solution containing the desired concentrationof hGH polymer prodrug, which subsequently was frozen and lyophilized.

RP-HPLC to detect free hGH: Mobile phase A was composed of 0.05% aqueousTFA (e.g. 0.5 mL in 999.5 mL HPLC-grade water) and mobile phase B wascomposed of 0.04% TFA in acetonitrile (e.g. 0.4 mL TFA in 999.6 mLacetonitrile). A Waters UPLC C18 BEH 300 Å 1.7 μm 2.1×50 mm column wasused. Flow rate was set to 0.4 mL/min, detection was at a wavelength of215 nm, the column running temperature was 30° C. (±5° C.). The samplecooler temperature was set at 4° C. and the sample injection load was100 μg (10 μL of a 10 mg/mL sample). Samples were transferred to 0.22-μmPVDF centrifugal filters (Millipore, # UFC30GVNB) and filtered bycentrifugation for 1 min at 9,000×g. Filtered samples were diluted to 10mg/mL with formulation buffer.

Tryptic digest of polymeric hGH prodrug and peptide mapping: Trypticdigestion of hGH-conjugates was performed at a trypsin/hGH ratio=1:20(w/w). The pH was adjusted with proteolysis buffer concentrate to pH=7.0to minimize the hGH-cleavage from the transient hGH-PEG conjugate duringthe digest. After 8 h incubation at 37° C. the digest was stopped by theaddition of 5% (w/w) phosphoric acid solution. The resulting peptidemixture was separated by RP-HPLC on a C18 column with 300 A pore sizeand 1.7 μm particle diameter. A gradient combination with 0.05 vol % aq.TFA and 0.04% TFA in acetonitrile as eluents and detection at 215 nm wascreated. The tryptic fragments which eluted with reasonable resolutionwere characterized via LC-MS and compared to theoretical monoisotopicmasses. The protein sequence coverage was 96% (184 of 191 amino acids).

Impurities were quantified as tryptic peptides based on their respectivepeak area relative to the peak area of the corresponding unmodifiedtryptic peptide. Under the conditions of the tryptic digest andsubsequent RP-HPLC-MS, both the succinimide intermediate and theisoaspartate could be quantified for ASP130 of the polymeric hGHprodrug.

Under the conditions of the tryptic digest and subsequent RP-HPLC-MS,aspartate formation resulting from ASN149 and ASN152 deamidation couldbe quantified for the polymeric hGH prodrug.

Under the conditions of the tryptic digest and subsequent RP-HPLC-MS,formation of the sulfoxide degradation product of MET14 could bequantified for the hGH polymer prodrug.

While this invention has been described in conjunction with the specificembodiments outlined above, it is evident that many alternatives,modifications, and variations will be apparent to those skilled in theart. Accordingly, the preferred embodiments of the invention as setforth above are intended to be illustrative, not limiting. Variouschanges may be made without departing from the spirit and scope of theinventions as defined in the following claims.

ABBREVIATIONS

-   LC-MS—liquid chromatography-coupled mass spectrometry-   PVDF—polyvinylidene fluoride-   RH—relative humidity-   RP-HPLC—reversed phase high performance liquid chromatography-   TFA—trifluoroacetic acid-   Tris—tris(hydroxymethyl)aminomethane-   UPLC—ultra performance liquid chromatography

The invention claimed is:
 1. A method of treating a disease which can betreated by recombinant human growth hormone (“rhGH”) in a patient, themethod comprising: administering to the patient a therapeuticallyeffective amount of a pharmaceutical composition comprising: 10-300mg/ml of rhGH polymer prodrug; 5-50 mM of succinic acid; 25-850 mg/ml oftrehalose dihydrate; wherein said pharmaceutical composition has a pHranging from 4.5 to 6; wherein the therapeutically effective amount ofsaid pharmaceutical composition is a single dose having a volume of notmore than 4 ml; and wherein within the rhGH polymer prodrug the rhGH istransiently linked via a reversible linker moiety to a polymer carrierand from which rhGH is released in its free form.
 2. The methodaccording to claim 1; wherein said disease is selected from the groupconsisting of growth hormone deficiency (GHD), adult onset growthhormone deficiency, Turner syndrome, Prader-Willi syndrome, short bowelsyndrome, chronic renal insufficiency, small for gestational age (SGA),AIDS wasting, anti-ageing, rheumatoid arthritis, idiopathic smallstature, short stature homeobox gene, somatopause, short statureassociated with prolonged steroid use, and Aarskog's syndrome.
 3. Themethod according to claim 2; wherein said disease is growth hormonedeficiency.
 4. The method according to claim 1; wherein the rhGH polymerprodrug has the chemical structure shown in (A):

wherein —HN-rhGH represents the rhGH residue attached to the reversiblelinker moiety; wherein R1, R2, R3, R4, and R5 are selected independentlyfrom the group consisting of hydrogen, methyl, ethyl, propyl, isopropyl,butyl, isobutyl, and tertiary butyl; wherein PEG represents a PEGylationresidue attached to the transient linker; wherein n=1 or 2; and whereinX is selected from C1 to C8 alkyl or C1 to C12 heteroalkyl.
 5. Themethod according to claim 4; wherein the moiety

 of formula (A) has the following structure:

wherein each c is independently an integer from 250 to
 750. 6. Themethod according to claim 1; wherein the rhGH polymer prodrug has thefollowing chemical structure:

wherein c is
 500. 7. The method according to claim 1; wherein the singledose of said pharmaceutical composition has a volume of 0.5 to 3.5 ml.8. The method according to claim 1; wherein the rhGH polymer prodrug issufficiently dosed in said pharmaceutical composition to providetherapeutically effective amount of rhGH for at least three days.
 9. Themethod according to claim 8; wherein the rhGH polymer prodrug issufficiently dosed in said pharmaceutical composition to providetherapeutically effective amount of rhGH for four days.
 10. The methodaccording to claim 9; wherein the rhGH polymer prodrug is sufficientlydosed in said pharmaceutical composition to provide therapeuticallyeffective amount of rhGH for five days.
 11. The method according toclaim 10; wherein the rhGH polymer prodrug is sufficiently dosed in saidpharmaceutical composition to provide therapeutically effective amountof rhGH for one week.
 12. The method according to claim 1; wherein saidpharmaceutical composition is comprised in a container.
 13. The methodaccording to claim 12; wherein said container is a dual-chamber syringe.14. The method according to claim 1; wherein said pharmaceuticalcomposition is administered by injection or infusion.
 15. The methodaccording to claim 14; wherein said pharmaceutical composition isadministered by injection.
 16. The method according to claim 1; whereinsaid pharmaceutical composition comprises: 10-300 mg/ml of the rhGHpolymer prodrug; 5-50 mM of the succinic acid; and 30-150 mg/ml of thetrehalose dihydrate.
 17. The method according to claim 16; wherein saidpharmaceutical composition comprises: 10-300 mg/ml of the rhGH polymerprodrug; 5-50 mM of the succinic acid; and 50-100 mg/ml of the trehalosedihydrate.
 18. The method according to claim 17; wherein saidpharmaceutical composition comprises: 30-60 mg/ml of the rhGH polymerprodrug; 10 mM of the succinic acid; and 70-85 mg/ml of the trehalosedihydrate; and wherein said pharmaceutical composition has a pH rangingfrom 4.5 to 5.5.
 19. The method according to claim 1; wherein saidpharmaceutical composition further comprises: at least one additionalbiologically active agent, either in its free form or as a prodrug,selected from the group consisting of IGF-1, ghrelin, ghrelin-likecompounds, gonadotropin releasing hormone agonists, gonadotropinreleasing hormone analogs, growth hormone releasing factors, growthhormone releasing analogs, gonadal steroids, antiandrogens,non-steroidal aromatase inhibitors, HIV combination therapy, free fattyacid regulators, anabolic steroids, estrogen agonists, estrogenantagonists, propranolol, appetite suppressants, osteoporosis drugs, andanti-diabetic drugs.
 20. The method according to claim 1; wherein saidpharmaceutical composition further comprises: one or more preservativeand/or antimicrobial.
 21. The method according to claim 1; wherein saidpharmaceutical composition further comprises: 0.7-1.1% (w/w)benzylalcohol.
 22. The method according to claim 1; wherein saidpharmaceutical composition further comprises: 0.2-0.4% (w/w) cresol.